102 general, been difficult to stimulate into mitosis or i m m u n o g l o b u l i n production. A l t h o u g h they possess E B V receptors they p r o d u c e little i m m u n o g l o b u l i n after stimulation with the virus, a n d cell lines have been very difficult to establish ~4. Differentiation to I g M a n d I g G production has b e e n reported to occur w h e n C L L cells are stimulated with antii m m u n o g l o b u l i n s in the presence of T-cell factors ~5. J o h n s t o n e argues that the production o f free light chain by C L L cells is a n o t h e r indication that these cells are i m m a t u r e B cells. T h e r e is certainly evidence that free light chain is secreted by B cells at a stage of differentiation probably corresponding to a n early B cell ~6. But free light chain is also produced by cells of the B lineage at a late stage of differentiation (i.e. p l a s m a cell), particularly in myelomatosis 17. H e n c e , free light chain is n o t a reliable differentiation stage marker. T h e r e are other m a r k e r s which, like the secretion of free light chain, are associated with both early a n d late stages of B-cell differentiation. C o m m o n A L L antigen a n d receptors for p e a n u t agglutinin are present on i m m a t u r e B cells as well as on cells of the post-antigen stage found in germinal centres Is. . T h e r e are some properties of C L L cells which cannot readily be a c c o m m o d a t e d within a simple 'frozen differentiation stage' hypothesis. T h e s e include the presence of avid Fc/~ receptors 19, the expression o f antigens shared by some T cellsz°'2~, a n d sensitivity to microtubular reagents 22, i n addition to the points J o h n s t o n e mentions. T h e r e is also good e v i d e n c e of a n a b n o r m a l i t y in the T l y m p h o c y t e subpopulation, in patients with B-cell C L L (Ref. 13). Finally, there is evidence that the growth fraction in C L L often resides in the l y m p h nodes 23, a n d in s o m e patients these dividing cells are f o u n d in clusters called proliferation centres 24, within l y m p h nodes. In contrast, the rate of lymphocyte proliferation w i t h i n the bone m a r r o w is 1ow23. •Early B-cell differentiation, o n the other h a n d , occurs in the bone m a r r o w with a possible period of m a t u r a t i o n in the spleen 25'26. T h e s e observations would be difficult to reconcile with the view that C L L cells represent a n intermediate stage of B-cell differentiation between pre-B cells a n d m a t u r e lymphocytes. T h e data available are insufficient to exclude the possibility that C L L cells represent B cells at a s o m e w h a t later stage of B-cell differentiation, as. suggested, for example, by Lukes a n d Collins 27. T h e fact that we are not able to accept s o m e ofJ o h n s t o n e ' s conclusions does not m e a n that we do not appreciate the value
Immunology Today, vol. 4, No. 4, 1983
of his s t i m u l a t i n g article. T h e r e is a t r e m e n d o u s a n d increasing a m o u n t of literature in this field which is in need of integration a n d interpretation. D. S. KUMARARATNE N. R. LING Department of Immunology, University of Binningham B15 2T,], UK.
References 1 I~randy, A., Fischer, A. and Friscelli, C. (1979)J. Immunol. 123, 2644 2 Jaafar, M. I. K. and Jefferis, R. (1982) Biochem. Soc. Trans. 10, 225 3 Partridge, L. J., Lowe, J., Hardie, D. L., Ling, N. R. and Jefferis, R. (1982)J. Immunol. 128, 1 4 Dhaliwal, H. S., Ling, N. R., Bishop, S. and Chapel, H. (1978) Clin. Exp. ImmunoL 31,226 5 de Gast, G. C. and Platts-Mills, T. A . E. (1979)J. Immunol. 122, 285 6 Stevenson, F. K., Hamblin, T.J., Stevenson, G. T. and Tutt, A. L. (1980)J. Exp. Med. 152, 1484 7 Stevenson, F. K., Hamblin, T. J. and Stevenson, G. T. (1981)J. Exp. Med. 154, 1965 8 Butcher, E. C., Rouse, R. V., Coffman, R. L., Nottenburg, C. N., Hardy, R. R. and Weissman, I. L. (1982)J~ ImmunoL 129, 2698 9 Benner, R., van Oudenaren, A., Bj6rklund, M., Ivars, F. and Holmberg, D. (1982) Immunol. Today 3, 243 10 Brown, N. A. and Miller, G. (1982)J. ImmunoL 128, 24 11 Morito, T., Bankhurst, A. D. and Williams, R. C. (1979)J. Clin. Invest. 64, 990 This letter was sent to Dr Johnstone, who replied as follows: SIR, I agree that we do not k n o w all the details of n o r m a l B-cell differentiation b u t it is still valid to consider C L L within the context of what is known. Such a process is two-way a n d we m a y even learn s o m e t h i n g a b o u t n o r m a l B lymphopoiesis from such a comparison. T h e old adage ' to u n d e r s t a n d the normal, first study the a b n o r m a l ' is as pertinent today (e.g. i m m u n o g l o b u l i n genes) as it was w h e n used to refer to metabolic m u t a n t s of bacteria. K u m a r a r a t n e a n d L i n g are correct to point out the problems of interpreting cell surface i m m u n o g l o b u l i n data a n d that is precisely w h y I relied mostly on measurem e n t o f total cell i m m u n o g l o b u l i n content or secreted i m m u n o g l o b u l i n in m y studies~. For example, K u m a r a r a t n e a n d L i n g report that the same proportion of B cells express surface IgG a n d IgA in n e w b o r n a n d adult blood, whereas the total cellular content of these isotypes clearly differs between neonates a n d adults (Table II in m y review). I n this context it is worth noting that cells can express i m m u n o g l o b u l i n isotypes other thar~ I g M a n d I g D on their surface without r e a r r a n g i n g D N A to delete the g
12 Rnuskanen, D., Pittard, III, W. B., Miller, K., Pierce, G., Sorensen, R. U. and Polmar, S. H. (1980)J. Immunol. 125, 411 13 Platsoucas, C. D., Galinski, M., Kempin, S., Reich, L., Clarkson, B. and Good, R. A. (1982)J. Immunol. 129, 2305 14 Rickinson, A. B., Finerty, S. and Epstein, M. A. (1982) Clin. Exp. Immunol. 50, 347 15 Yoshizaka, K., Nakagawa, T., Kaieda, T., Muraguchi, A., Yamamura, Y. and Kishomoto, T. (1982)J. Immunel. 128, 1296 16 Hannam-Harris, A. C., Gordon, J. and Smith, J. L. (1980)J. Immunol. 125, 2177 17 Gahon, D. A. G. and MacLennan, I. C. M. (1982) Clinics in Hnen~tology 11, 561 18 Stein, H. and Mason, D. W. (1982) Clinics in Haematology 11, 53 19 Pickler, W. J. and Knapp, W. (1977)J. Immunol. 118, 1010 20 Martin, P. J. (1980)Immunogenetics(NY) 11, 429 21 Boumsell, L., Coppin, H., Pham, D., Taynal, B., Lemerle, J., Dansset, J. and Bernard, A. (1980)J. Exp. Med. 152, 229 22 Sehrek, R. (1975) Br. J. Exp. Pathol. 56, 280 23 Trepel, F. and Schick, P. (1976) in Hamatologie und Blut Transfusion, Bd 18 (Loeffier, H., ed.), pp. 33-47, Lehmans, Munich 24 Lennert, K. (1978) in Malignant Lymphomas Other Than Hodgkin's Disease, p. 110, SpringerVerlag, Berlin 25 Osmond, D. G. (1979) in B Lymphocytes in the Immune Response (Cooper, M. D., Mosier, D. E., Scher, I. and Vitetta, E. S., eds), pp. 63-70, Elsevier North-Holland, New York 26 Strober, S. (1976) in Immunoaspectsof the Spleen (Battisto, J. R. and Streilin, J. N., eds), p. 129, Elsevier North-Holland, Amsterdam 27 Lukes, R.J. and CoSins, R. D. (1975) Br. J. Cancer 31, Suppl. II, 1 gene 2, p r e s u m a b l y by a n R N A splicing process, thus allowing t h e m to simultaneously or subsequently synthesize IgM. In m y review, I covered the secretion of small a m o u n t s of intact I g M a n d I g D by unstimulated CLL cells which K u m a r a r a t n e a n d L i n g point out, a n d to m y knowledge no one has s h o w n that such secretion is inconsistent with i m m a t u r i t y . Similarly, the secretion of excess light chain, although not definite proof of i m m a t u r i t y , is certainly consistent with it. T h e relationship between m a t u r i t y and the immunoglobulin isotype produced after in-vitro stimulation was discussed at s o m e length in m y review, b u t I would like to point out one very recent s t u d y 3 which clearly shows a m u c h higher ratio of I g M : I g G secreted followi n g E B V stimulation of cells f r o m foetal liver (104) a n d cord blood (62) c o m p a r e d with adult blood (2.6). I discussed the presence of s o m e 'T-cell-specific' antigens on C L L cells, their e x t r e m e sensitivity to the microt u b u l a r reagent colchicine a n d the abnormalities in T cells from C L L patients, which K u m a r a r a t n e a n d L i n g raise. Indeed the m o s t recent evidence on the last point c o m e s from m y own laboratory *
Immunology Today, vol. 4, No. 4, 1983
and suggests that T cellsin the circulation of some CLL patients may also be immature. In attempting to make sense of the mass of data on CLL, some of it conflicting, which has accumulated over the years, inevitablysome pieces of evidence or individual cases have to be put to one side in order to derive a hypothesis that accommodates most of the facts. By this process, I conclude that C LL cellsare certainly not equivalent to mature, circu-
103
lating B lymphocytes (as had become dogma in many scientificcircles) and that the balance of evidence indicates that they represent a more immature stage. Whether they are identical to a normal immature stage is for future study. A. P. J O H N S T O N E
Department of Immunology, St George's Hospital Medical School, London S W 1 7 ORE, UK.
References 1 Johnstone, A. P., Jensenius, J. C., MiHard, R. E. and Hudson, L. (1982) Clin. Exp. Immunol. 47, 697-705 2
Yaoita, Y., Kumagai, Y., Okumura, K. and Honjo, T. (1982) Nature (Lo~lon) 297, 697-699
3
Pereira, S., Webster, D. and Platts-Mills, T. (1982) Eur. J. Immunol. 12, 540-546
4 Johnstone, A. P., Height, S. and Millard, R. E. (1982) Biosci. Rep. 2, 535-542
Phase variations in the modulation of the immune response Martin Davies Stimulation and depression may be two sides of the same coin of immunomodulation. HereMartin Davies discusses afeature of the u~te of adjuvants, he and others have observed.
Immunopotentiators, or adjuvants, were originally described as agents that were able to heighten immune responses to antigenic material or to stimulate immune responses to material that was apparently non-immunogenic. By this definition it was assumed that if doseresponse relationships were established by injecting animals with a constant antigen dose, coupled with variable quantities of the adjuvant, a sigmoid curve of potentiation would be obtained. However, it became apparent from the work on endotoxin from Gram-negative bacteria that increased immune reactivity was only observed following intervention with low doses of endotoxin~'2 and that by increasing the dose, the potentiation of the response decreased until at very high doses positive suppression was encountered~. Further, with the 1959 report 4 on the use of Bacille Calmette-Gu~rin (BCG) in the reduction of tumour incidence in A K R mice, enormous interest was stimulated in the use of adjuvants, especially in the field of cancer immunotherapy. However, these studies, far from resolving the stimulation/suppression problem and providing a rational basis for effective adjuvant therapy for cancer, have yielded similar conflicting reports in that some authors have reported BCG to be immuno stimulatory*, others that it is immunodepressive5'6 and others that it exerted no effect at alF. Hence, BCG could exhibit effects on the immune system that showed a phase variation and could either be positive (stimulatory) or negative (depressive). The contrasting effects, although similar to the pattern previously reported for endotoxin, were usually obtained within a smaller dose range. Since different routes and schedules of BCG administration were employed, it is difficult to compare these stimulatory and depressive effects directly. How-
ever, examination of the literature has revealed that if potential immunoadjuvants (e.g. BCG, Co~nebaclerium parvum, endotoxin, vitamin E, etc.) were investigated by using the adjuvant dose as a continuous variable (all other parameters being constant), then the immunostimulatory/immunodepressive (or phase variation) effect could be obtained within a single experiment and within a narrow dose range, provided that the dose interval was small enough. In such cases there was a marked absence of a 'traditional' dose-response relationship and phase variations were evident. This phasic phenomenon and the erratic effect of BCG on the survival of A K R mice with spontaneous leukaemia led Ungaro, Drake and Mardineya to comment that ' . . . a marked difference in outcome of treatment can depend on the dose used. Conflicting reports about the efficacy of BCG in other tumour systems may be due partially to an incorrect selection of dose.' Further, in some of the reports where sufficient dose points were studied, instead of a biphasic response, a multiphase phenomenon was detected, which exhibited several regions of stimulation and several of depression over a comparatively small dose range. Examples of the phase variation effect can be found throughout the literature, although, in general, there are no specific references since the effect has not so far been studied as a phenomenon in its own right. However, such a phenomenon is not only important for understanding how immunopotentiators work (or do not work), but its appreciation could lead to a better understanding of how and when to use such agents in the immunotherapy of cancer.
Institute'ofGenetics, Universityof Glasgow, Glasgow G 11 5JS, U.K.
The phase variation phenomenon can best be defined as a lack of a 'true' dose-response relationship between
Definition and description of the phase variation phenomenon © 1983,El~vierSciencePublishersB,V,,Amsterd~ 0167 4919/83/$02.00
Immunology Today, vol. 4, No. 4, 1983
of his s t i m u l a t i n g article. T h e r e is a t r e m e n d o u s a n d increasing a m o u n t of literature in this field which is in need of integration a n d interpretation. D. S. KUMARARATNE N. R. LING Department of Immunology, University of Binningham B15 2T,], UK.
References 1 I~randy, A., Fischer, A. and Friscelli, C. (1979)J. Immunol. 123, 2644 2 Jaafar, M. I. K. and Jefferis, R. (1982) Biochem. Soc. Trans. 10, 225 3 Partridge, L. J., Lowe, J., Hardie, D. L., Ling, N. R. and Jefferis, R. (1982)J. Immunol. 128, 1 4 Dhaliwal, H. S., Ling, N. R., Bishop, S. and Chapel, H. (1978) Clin. Exp. ImmunoL 31,226 5 de Gast, G. C. and Platts-Mills, T. A . E. (1979)J. Immunol. 122, 285 6 Stevenson, F. K., Hamblin, T.J., Stevenson, G. T. and Tutt, A. L. (1980)J. Exp. Med. 152, 1484 7 Stevenson, F. K., Hamblin, T. J. and Stevenson, G. T. (1981)J. Exp. Med. 154, 1965 8 Butcher, E. C., Rouse, R. V., Coffman, R. L., Nottenburg, C. N., Hardy, R. R. and Weissman, I. L. (1982)J~ ImmunoL 129, 2698 9 Benner, R., van Oudenaren, A., Bj6rklund, M., Ivars, F. and Holmberg, D. (1982) Immunol. Today 3, 243 10 Brown, N. A. and Miller, G. (1982)J. ImmunoL 128, 24 11 Morito, T., Bankhurst, A. D. and Williams, R. C. (1979)J. Clin. Invest. 64, 990 This letter was sent to Dr Johnstone, who replied as follows: SIR, I agree that we do not k n o w all the details of n o r m a l B-cell differentiation b u t it is still valid to consider C L L within the context of what is known. Such a process is two-way a n d we m a y even learn s o m e t h i n g a b o u t n o r m a l B lymphopoiesis from such a comparison. T h e old adage ' to u n d e r s t a n d the normal, first study the a b n o r m a l ' is as pertinent today (e.g. i m m u n o g l o b u l i n genes) as it was w h e n used to refer to metabolic m u t a n t s of bacteria. K u m a r a r a t n e a n d L i n g are correct to point out the problems of interpreting cell surface i m m u n o g l o b u l i n data a n d that is precisely w h y I relied mostly on measurem e n t o f total cell i m m u n o g l o b u l i n content or secreted i m m u n o g l o b u l i n in m y studies~. For example, K u m a r a r a t n e a n d L i n g report that the same proportion of B cells express surface IgG a n d IgA in n e w b o r n a n d adult blood, whereas the total cellular content of these isotypes clearly differs between neonates a n d adults (Table II in m y review). I n this context it is worth noting that cells can express i m m u n o g l o b u l i n isotypes other thar~ I g M a n d I g D on their surface without r e a r r a n g i n g D N A to delete the g
12 Rnuskanen, D., Pittard, III, W. B., Miller, K., Pierce, G., Sorensen, R. U. and Polmar, S. H. (1980)J. Immunol. 125, 411 13 Platsoucas, C. D., Galinski, M., Kempin, S., Reich, L., Clarkson, B. and Good, R. A. (1982)J. Immunol. 129, 2305 14 Rickinson, A. B., Finerty, S. and Epstein, M. A. (1982) Clin. Exp. Immunol. 50, 347 15 Yoshizaka, K., Nakagawa, T., Kaieda, T., Muraguchi, A., Yamamura, Y. and Kishomoto, T. (1982)J. Immunel. 128, 1296 16 Hannam-Harris, A. C., Gordon, J. and Smith, J. L. (1980)J. Immunol. 125, 2177 17 Gahon, D. A. G. and MacLennan, I. C. M. (1982) Clinics in Hnen~tology 11, 561 18 Stein, H. and Mason, D. W. (1982) Clinics in Haematology 11, 53 19 Pickler, W. J. and Knapp, W. (1977)J. Immunol. 118, 1010 20 Martin, P. J. (1980)Immunogenetics(NY) 11, 429 21 Boumsell, L., Coppin, H., Pham, D., Taynal, B., Lemerle, J., Dansset, J. and Bernard, A. (1980)J. Exp. Med. 152, 229 22 Sehrek, R. (1975) Br. J. Exp. Pathol. 56, 280 23 Trepel, F. and Schick, P. (1976) in Hamatologie und Blut Transfusion, Bd 18 (Loeffier, H., ed.), pp. 33-47, Lehmans, Munich 24 Lennert, K. (1978) in Malignant Lymphomas Other Than Hodgkin's Disease, p. 110, SpringerVerlag, Berlin 25 Osmond, D. G. (1979) in B Lymphocytes in the Immune Response (Cooper, M. D., Mosier, D. E., Scher, I. and Vitetta, E. S., eds), pp. 63-70, Elsevier North-Holland, New York 26 Strober, S. (1976) in Immunoaspectsof the Spleen (Battisto, J. R. and Streilin, J. N., eds), p. 129, Elsevier North-Holland, Amsterdam 27 Lukes, R.J. and CoSins, R. D. (1975) Br. J. Cancer 31, Suppl. II, 1 gene 2, p r e s u m a b l y by a n R N A splicing process, thus allowing t h e m to simultaneously or subsequently synthesize IgM. In m y review, I covered the secretion of small a m o u n t s of intact I g M a n d I g D by unstimulated CLL cells which K u m a r a r a t n e a n d L i n g point out, a n d to m y knowledge no one has s h o w n that such secretion is inconsistent with i m m a t u r i t y . Similarly, the secretion of excess light chain, although not definite proof of i m m a t u r i t y , is certainly consistent with it. T h e relationship between m a t u r i t y and the immunoglobulin isotype produced after in-vitro stimulation was discussed at s o m e length in m y review, b u t I would like to point out one very recent s t u d y 3 which clearly shows a m u c h higher ratio of I g M : I g G secreted followi n g E B V stimulation of cells f r o m foetal liver (104) a n d cord blood (62) c o m p a r e d with adult blood (2.6). I discussed the presence of s o m e 'T-cell-specific' antigens on C L L cells, their e x t r e m e sensitivity to the microt u b u l a r reagent colchicine a n d the abnormalities in T cells from C L L patients, which K u m a r a r a t n e a n d L i n g raise. Indeed the m o s t recent evidence on the last point c o m e s from m y own laboratory *
Immunology Today, vol. 4, No. 4, 1983
and suggests that T cellsin the circulation of some CLL patients may also be immature. In attempting to make sense of the mass of data on CLL, some of it conflicting, which has accumulated over the years, inevitablysome pieces of evidence or individual cases have to be put to one side in order to derive a hypothesis that accommodates most of the facts. By this process, I conclude that C LL cellsare certainly not equivalent to mature, circu-
103
lating B lymphocytes (as had become dogma in many scientificcircles) and that the balance of evidence indicates that they represent a more immature stage. Whether they are identical to a normal immature stage is for future study. A. P. J O H N S T O N E
Department of Immunology, St George's Hospital Medical School, London S W 1 7 ORE, UK.
References 1 Johnstone, A. P., Jensenius, J. C., MiHard, R. E. and Hudson, L. (1982) Clin. Exp. Immunol. 47, 697-705 2
Yaoita, Y., Kumagai, Y., Okumura, K. and Honjo, T. (1982) Nature (Lo~lon) 297, 697-699
3
Pereira, S., Webster, D. and Platts-Mills, T. (1982) Eur. J. Immunol. 12, 540-546
4 Johnstone, A. P., Height, S. and Millard, R. E. (1982) Biosci. Rep. 2, 535-542
Phase variations in the modulation of the immune response Martin Davies Stimulation and depression may be two sides of the same coin of immunomodulation. HereMartin Davies discusses afeature of the u~te of adjuvants, he and others have observed.
Immunopotentiators, or adjuvants, were originally described as agents that were able to heighten immune responses to antigenic material or to stimulate immune responses to material that was apparently non-immunogenic. By this definition it was assumed that if doseresponse relationships were established by injecting animals with a constant antigen dose, coupled with variable quantities of the adjuvant, a sigmoid curve of potentiation would be obtained. However, it became apparent from the work on endotoxin from Gram-negative bacteria that increased immune reactivity was only observed following intervention with low doses of endotoxin~'2 and that by increasing the dose, the potentiation of the response decreased until at very high doses positive suppression was encountered~. Further, with the 1959 report 4 on the use of Bacille Calmette-Gu~rin (BCG) in the reduction of tumour incidence in A K R mice, enormous interest was stimulated in the use of adjuvants, especially in the field of cancer immunotherapy. However, these studies, far from resolving the stimulation/suppression problem and providing a rational basis for effective adjuvant therapy for cancer, have yielded similar conflicting reports in that some authors have reported BCG to be immuno stimulatory*, others that it is immunodepressive5'6 and others that it exerted no effect at alF. Hence, BCG could exhibit effects on the immune system that showed a phase variation and could either be positive (stimulatory) or negative (depressive). The contrasting effects, although similar to the pattern previously reported for endotoxin, were usually obtained within a smaller dose range. Since different routes and schedules of BCG administration were employed, it is difficult to compare these stimulatory and depressive effects directly. How-
ever, examination of the literature has revealed that if potential immunoadjuvants (e.g. BCG, Co~nebaclerium parvum, endotoxin, vitamin E, etc.) were investigated by using the adjuvant dose as a continuous variable (all other parameters being constant), then the immunostimulatory/immunodepressive (or phase variation) effect could be obtained within a single experiment and within a narrow dose range, provided that the dose interval was small enough. In such cases there was a marked absence of a 'traditional' dose-response relationship and phase variations were evident. This phasic phenomenon and the erratic effect of BCG on the survival of A K R mice with spontaneous leukaemia led Ungaro, Drake and Mardineya to comment that ' . . . a marked difference in outcome of treatment can depend on the dose used. Conflicting reports about the efficacy of BCG in other tumour systems may be due partially to an incorrect selection of dose.' Further, in some of the reports where sufficient dose points were studied, instead of a biphasic response, a multiphase phenomenon was detected, which exhibited several regions of stimulation and several of depression over a comparatively small dose range. Examples of the phase variation effect can be found throughout the literature, although, in general, there are no specific references since the effect has not so far been studied as a phenomenon in its own right. However, such a phenomenon is not only important for understanding how immunopotentiators work (or do not work), but its appreciation could lead to a better understanding of how and when to use such agents in the immunotherapy of cancer.
Institute'ofGenetics, Universityof Glasgow, Glasgow G 11 5JS, U.K.
The phase variation phenomenon can best be defined as a lack of a 'true' dose-response relationship between
Definition and description of the phase variation phenomenon © 1983,El~vierSciencePublishersB,V,,Amsterd~ 0167 4919/83/$02.00
