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-


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


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 Phase variation effect with agents of bacterial






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~CG DOSE ~Jg) Fig. 1. The effect of a series of BCG doses on the growth of LMC 1 tumottr in inbred Lewis rats:

Various BCG doses (200-2000 ~g) were injected intravenously into Lewis rats 12 days before the subcutaneous implant of a lethal dose of 5 x 105LMC l tumourceUs.The BCG dose is plotted against thetumour size ( • ) 50 days after implant and against the percent animal survival ([2])3 months after implant. (Each point represents the mean +SE of 5 tumours.) There was a biphasic pattern over the dose range 200-2000 ~g of freeze-dried vaccine. Two regions of stimulation were separated by a region of marked depression, stimulation being defined by decreased tumour growth and enhanced host survival, and depression by the opposite effects. From other studiesl° it was assumed that these effects had an immunologicalbasis. At the peak of stimulation (250 pg dose of BCG) tumour growth was reduced from 2300 mm2 to 600 mm2 and host survival increased from 0% to 70%. Beyond doses of 250 ~g there was depression, at its maximum increasing tumour growth from 2300 mm2 to 3800 mm2 and an increasing mortality rate to 100%. Beyond doses of 750 8g there was again a beneficial effect, with 2000 ~g decreasing tumour growth to 900 mm2 and increasing host survival to 60%.

increasing doses of the adjuvant a n d their effect on the i m m u n e response against a specific antigenic challenge. The p h e n o m e n o n is characterized by the observation that small changes in the adjuvant dose result in a phase change in the i m m u n e response - i.e. the response changes from being stimulated (positive) to being depressed (negative), compared with the response obtained in the absence of adjuvant. Further, since the dose is able to act as a continuous variable, i.e. it is possible to obtain different degrees of stimulation a n d depression, then it should also be possible to choose a dose that exerts no effect at all. Fig. 1 shows an example of the phase variation effect in t u m o u r responses. T h e results obtained in the experiments were initially disturbing as there was apparently no 'proper' dose-response relationship. However, the phase change was taken to be a real effect a n d the literature was examined for further examples employing otl~er agents~s well as BCG.

origin T h e first few examples concern the use of B C G vaccine. U s i n g a n allogeneic B6AFI mouse mastocytoma P815-X2 system, Davies a n d Sabbadini 1°observed the effect on cellular cytotoxicity of various doses of B C G vaccine r a n g i n g from 200 to 2000/Jg per animal. B C G was administered intravenously 12 days before the t u m o u r implant a n d the splenic cellular activity, expressed as lytic units (LU) per spleen, was measured 15 days after this implant. T h e tumour-alone controls exhibited 200 L U / s p l e e n a n d the introduction of B C G increased this response so that 500 /~g B C G potentiated the splenic activity to 2200 LU/spleen. However, as the B C G dose was increased, the response, although still potentiated, declined to 900 LU/spleen at the 1000/ag level. F u r t h e r increases in B C G resulted in a second stimulatory region, and 1500/ag B C G stimulated 2500 LU/spleen. Although the experiment was continued at only a few dose points beyond 1500/~g, it appeared that the response was b e g i n n i n g to decline to form a second region of depression. This experiment was performed repeatedly a n d it was apparent that the biphasic pattern was found in only 30% of the animals tested. Over the same B C G dose range, the remainder of the animals exhibited a pattern that showed a single peak of stimulation, followed by a decline in response. Although this difference could indicate a shift to the right in the sensitivity of the phasic effect, it was subsequently found that the cellular cytotoxic response in those animals showing only a single peak was mediated solely by T cells, but in those animals that exhibited the biphasic variation the response was mediated by both T cells a n d macrophages. Studies with the Lewis rat/LMC~ system, described earlier, in which a live t u m o u r vaccine was used in combination with B C G " , showed that the dose of B C G chosen could profoundly affect the growth of a challenge tumour. For example, d u r i n g the first 4 weeks after i m p l a n t in control animals the t u m o u r grew at a rate of 90 mm2/ week. However, with the introduction of B C G (coupled with a constant t u m o u r vaccine) the growth rate varied considerably depending o n the B C G dose. Hence, the rate was decreased to 23 mm2/week with 125/ag BCG, b u t rose to 61 mm2/week (although still below control values) with 500/~g. This was followed by a decrease to 41 mm2/ week with 1000 /~g and a subsequent increase to 90 mm2/week with 2000/~g. T h u s , despite the presence of the t u m o u r vaccine which provided some protection, the phase variation effects of the B C G could still be observed. Other workers using B C G vaccine have obtained a similar pattern of results. Moore, Lawrence and Witherow 12 observed the effects of various doses of B C G on the survival of AS rats challenged with the osteosarcoma CC5, and found that significant protection, 75 % a n d 100 % survival, was obtained with 0.16 x 106 a n d 2.5 x 10~ organisms respectively. However, between these two peaks was a n area of reduced effectiveness, with 0.63 x 106 B C G organisms showing only 25 % survival of the hosts. There is also a biphasic effect of B C G on A K R mouse survival with spontaneous leukaemia8 a n d a similar effect on h a e m a g g l u t i n i n production in C F D H guinea-pigs challenged with sheep red cells Is.

Immunology Today, vol. 4, No. 4, 1983

A biphasic effect has also been reported with C. parvum intervention in B6D2F1 mice challenged with P815 ~4. The effect was real but not very marked which was possibly a reflection of the dose intervals chosen. Similarly, Bober, Kranepool and HollandeP 5 found that the protection afforded by Escherichia coli endotoxin of BALB/c mice challenged with plasmacytoma M O P C 315 did not appear as a 'true' dose-response relationship, leading the authors to comment that they were ' . . . particularly disturbed by the lack of a dose response to endotoxin.' They attributed this to endogenous endotoxin and to the contamination of reagents with minute amounts ofendotoxin which effectively concealed a proper dose response.

Phase variation effect with other agents Besides agents of bacterial origin, phase variations have been reported, with agents as diverse as coenzyme Oo0, zymosan, poly A: poly U and vitamin E. Bliznakov 16 has observed that 50 /~g and 100 /~g of coenzyme Q~0 provided significant protection (45% and 56% survival respectively) of Swiss Webster mice challenged with feline leukaemia virus-induced leukaemia, while 75/~g and 125 /~g provided only minimal protection with 8% and 19% survival respectively. Bradner, Clarke and S~ock ~7 reporting a 'broad spectrum response' with zymosan, the dose response showing ' . . . no suggestion of a straight line function'. The zymosan was administered on a per kilogram body weight basis to Swiss Albino mice implanted subcutaneously with a lethal dose of Sarcc~ma 180. The 20 mg and 160 mg doses provided significant protection of the animals and resulted in a 60% and 62% survival rate respectively. However, the intermediate dose, 40 mg, showed only 32% animal survival, while the larger dose of 320 mg also reduced survival to 25 %. IfDBA/1 mouse spleens were cultured in the presence of sheep red cells, then the number of resulting plaque forming cells could be enhanced in the presence of the double-stranded polyribonucleotide, poly A: poly U 18. The addition of various log doses of the poly A: poly U revealed a dose response, which exhibited a non-linear relationship over the range 0.1-10 /~g. Finally, Heinzerling, Tengerdy, Wick and Leuker 19 showed that vitamin E could affect the survival of Swiss Webster mice infected with Diplococcus pneumoniae if administered as a food additive. The authors noted that a biphasic pattern resulted if different amounts of the vitamin were used. The 180 nag per kilogram of food dose provided 85% protection while the next dose level tested, 240 rag, reduced this protection to 40%. However, the final dose tested, 360 rag, again increased the survival to 65% to produce a biphasic pattern.

Conclusions, speculations and implications The phenomenon of phase variation is apparent with a variety of agents that are known to modulate immune reactivity against antigenic stimuli. Since the phenomenon has not been fully studied (or firmly established) as an entity in itself, examples are often difficult to find in the literature. When dose-response relationships are investigated the dose interval chosen is

105 often too large to demonstrate effectively the phenomenon. In the examples cited above the effect appears to be both real and universal, affecting a range of immune responses. Some of the examples are not well defined ~3'14'~ while others appear quite convincing~-~2'~5la.19.In general, small changes in dose produce profound changes in the response. The reason why the phenomenon is evident under certain conditions is difficult to ascertain, as is the source of the variation - whether it is inherent in the host or in the adjuvant or in fact results from the interaction of the two. In the case of BCG, it has been suggested that variations in both the strain of the organism and in the particular batch of vaccine can result in irreproducible and erratic results 2°'21. However, this source of variation would not account for the observed data since the various doses of BCG vaccine can act as a proper continuous variable. There are graded increases in the levels of both stimulation and depression, which would suggest something more than just a random source of error built into the vaccine. Bober, Kranepool and Hollander 15suggested that the variable responses to E. coli endotoxin were due to the presence of endogenous endotoxin or endotoxin contamination of reagents, which superimposed their own dose response on that of the E. coli endotoxin under investigation. However this would imply that if the phase variation effect was consistently obtained and a proper dose response to E. coli endotoxin was expected, then the contamination itself, which was also able to affect immune reactivity, would be responsible for the phase changes, and therefore such contamination would not be a randomized event. Hence, if the phase effect was an artefact of contamination or an 'in-built random variation' in the adjuvant source, the effect would be truly random and not so well ordered, and certainly not amenable to so many independent but similar observations. The effect does not seem to be an in-vitro artefact of the assay systems used to detect the immune response because tumour growth and/or host survival were the test criteria used in some examples. There is no indication at present of what the mechanism of the effect might be. If the effector cells of the immune system, or the helper cells that control them were divided into numerous subpopulations with different thresholds of responsiveness to the adjuvant some might respond to low and intermediate doses of the adjuvant, while others might be 'switched on' or alternatively 'switched off" at the higher levels. Phase variation might then be a composite effect made up by superimposing the stimulation and subsequent decline in the activity of the different subpopulations. Another mechanism might involve the differential stimulation of different types of effectors 1°. For example, as quoted earlier, in the 30% of B6AF mice which showed a biphasic pattern of stimulation of cellular immunity on challenge with P815, the response was mediated by both T cells and macrophages while in the remainder, where a single peak of stimulation was followed by depression over the same BCG dose range, the response was mediated solely by T cells. If the mechanisms of phase effects are speculative, their implications are wide ranging, especially in the field of cancer immunotherapy. A small change in the dose of the therapeutic agent can result in the negation of a


Immunology Today, vol. 4, No. 4, 1983

potentially beneficial effect or, in extreme circumstances, can benefit the tumour. It is thus essential that doseresponse relationships are firmly established and understood. Selection of the correct dose of adjuvant for therapy will not solve all the problems, but attention to this fact should help in defining the true limitation of the therapy treatment. The present limitations of the strategy are those imposed by the use of suboptimal conditions for the administration of the immunotherapeutic agent.

References 1 Condie, 17,. M., Zak, S.J. and Good, R. A. (1955)Fed. Proc. 14, 459-460 2 Johnson, A. G., Gaines, S. and Landy, M. (1956),]. Exp. Med. 103, 225-246 3 Bradley, S. G. and Watson, D. W. (1964) Proc. Soc. Exp. Biol. 117, 570-572 4 Old, L. J., Clarke, D. A. and Benacerraf, B. (1959) Nature(London) 184, 291-292 5 Chee, D. O. and Bodurtha, A. J. (1974) Int. J. Cancer 14, 137-144

6 Piessens, W. F., Lachapelle, F. L., Legros, N. and Heuson, J.-C. (1970) Nature (London) 228, 1210 7 Ziegler, J. L. and Magrath, I. T. (1973) Natl Cancer Inst. Moncgr. 39, 199-202 8 Ungaro, P. C., Drake, W. P. and Mardiney, M. R. (1973),]. Natl Cancer Inst. 50, 125-128 9 Davies, M. and Sabbadini, E. (1978)J. Nail Cancer Inst. 60, 1069-1073 10 Davies, M. and Sabbadini, E. (1978),]. Natl Cancer Inst. 60, 1059-1068 11 Davies, M. and Sabbadini, E. (1979) Cancer Res. 39, 959-965 12 Moore, M., Lawrence, N. and Witherow, P. J. (1974) Eur. J. Cancer 10, 673-682 13 Peters, L. C., Hanna, M. G., Gutterman, J. V., Mavligit, G. M. and Hersh, E. M. (1974) Proc. Soc. E.xp. Biol. Med. 147, 344-349 14 Scott, M. T. (1974),]. Nail Cancer Inst. 53, 861-865 15 Bober, L. A., Kranepool, M.J. and Hollander, V. P. (1976) Cancer Res. 36, 927-929 16 Bliznakov, E. G. (1973) Pr0c. NatlAcad. Sd. U.S.A. 70, 390-394 17 Bradner, W. T., Clarke, D. A. and Stock, C. (3. (1958) Cancer Res. 18, 347-351 18 Jaroslaw, B. N. and Ortiz-Ortiz, L. (1972) Cell. Immunol. 3, 123-132 19 Heinzerling, R. H., Tengerdy, R. P., Wick, L. L. and Leuker, D. (3. (1974) Infect. Immun. 10, 1292-1295 20 Willmott, N., Pirara, M. V. and Baldwin, R. W. (1979)J. Natl Cancer Inst. 63, 787-796 21 Mokyr, B. B., Bennett, J. A., Braun, D. P., Hengat, J. C. D., Mitchell, M. S. and Dray, S. (1980),]. Natl Cancer Inst. 64, 339-344

Mutant monoclonal antibodies In a recent article Yelton and ScharfP tried to answer the question of whether mutant monoclonal antibodies could replace chemical methods of altering antibody molecules. As an example they mentioned the production of mouse Fab molecules by mutant hybridoma lines, as an alternative to the conventional means of making the fragments by proteolytic cleavage of the protein. Fab molecules would be preferred in assays where the Fc portion of antibodies might bind non-specifically or via Fc receptors to non-target cells. The authors did not forget the topical issues of the possible therapeutic use of monoclonal antibodies, where again the (this time human) Fc portion might cause non-specific effects. Mutant antibodies made by hybridomas have the advantage of a never-ending source. The desired property, such as the inability to bind to Fc receptors or to fLx complement, could be engineered through subtle changes of the molecule (best point mutations) rather than chemical methods. The charm of the paper by Yelton and Scharff is that they did not make three papers out of the work they have done: the derivation of mutant lines of an antiarsonyl specific IgG2b-secreting hybridoma; the generation of rat monoclonal antibodies against each of the three mouse constant Y2bdomains; and the testing of the mutant antibodies' biological functions. They describe antibodies lacking most of the third domain, which are predominantly secreted as hemimers, are deficient in protein A binding and Fc receptor binding, but still activate complement though only via the minor dimeric antibody population. Another class of variant molecules have lost most of the CH2 domain; they are secreted as dimers, do © 1983,ElsevierSciencePublishersB,V.,Amsterdam 0167- 4919/83/$01.00

not bind protein A or to Fc receptors and do not activate complement. The usefulness of the rat rnonoclonal anti Y2b antibodies (besides their potential in mapping new mutants) was demonstrated by showing that a particular antiy2b antibody could not be inhibited by either the CH2 or the Cu3 loss variants. It is therefore suggested that the antibody binds to the Cn2-3 boundary, which is well in accord with its potential to inhibit protein A binding to the parental Y2b molecule. Monoclonal antibodies thus map more precisely than whole domain losses the site of interaction of the antibody with some effector molecules. A very similar series of mutant IgM-secreting hybridoma lines has been generated by Shulman, K6hler and collaborators z3. They have derived anti-trinltrophenyl- or anti-phosphorylcholine-binding IgM variants lacking C/al, C/al and 2, C/al, 2 and 3 or various parts of the C terminal portion of the/~ chain. The variants were used to map a series of rat monoclonal antibodies, and eight antigenic determinants - two for each of the four C/a domains - were identified (Potash and Leptin, unpublished observations). In these studies the aim of deriving variants with more subtle changes which alter function was achieved 2. Pentameric IgM of normal size and normal antigenic binding capacity was isolated which was unable to activate complement. The comparison of the structure of the parental and mutant/~ chain may give some insights into complement-IgM interactions. The generation of mutant antibody-secreting cell lines will therefore be very fruitful in elucidating structurefunction relationships of antibody molecules. But will the

Phase variations in the modulation of the immune response.

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