Immunology 1979 38 481 Suppressed or enhanced antibody responses in vitro after BCG treatment of mice: importance of BCG viability CAROLYN A. BROWN, I. N. BROWN* & V. S. SLJIVIC Departments of Immunology and *Bacteriology, St Mary's Hospital Medical School, London Acceptedfor publication 24 May 1979 Summary. Mycobacterium bovis, BCG, is known to be capable of either enhancing or suppressing various immune responses. Using a standard technique and number of organisms, some of the parameters pre- determining whether enhancement or suppression will occur have been investigated. Dead BCG given intra- venously into mice caused an enhancement of the antibody response in vitro to sheep erythrocytes. In contrast, the same number of viable organisms caused suppression if given intravenously but enhancement if given subcutaneously. The inclusion of 25% or more killed organisms in an intravenous inoculum of fully viable organisms changed suppression to enhance- ment. Treatment of BCG infected mice with strepto- mycin lessened the suppression but did not change it to enhancement. The possible causes of suppression are discussed. INTRODUCTION Mycobacterium bovis BCG is an effective immuno- prophylactic for tuberculosis and is known to have a range of effects, from inhibition to potentiation, on various other immune responses. Attempts have Correspondence: Dr C. A. Brown, Department of Immunology, St Mary's Hospital Medical School, London W2 IPG. 0019-2805/79/1100-0481$02.00 C) 1979 Blackwell Scientific Publications already been made to exploit its immunopotentiating activity for tumour immunotherapy (Baldwin & Pimm, 1978) and for the prevention of leprosy (Webb, Mims & Turk, 1979) but with equivocal results. With few exceptions, the assumption has been made that BCG preparations which elicit an immunity effective against tubercle bacilli will also be those most effective in the induction of anti-tumour and anti-leprosy im- munity. This may not be so. Although a wide literature on the biological activities of BCG is developing, its mode of action is still not fully understood. Compari- son of published results is difficult because (a) several substrains of BCG with known differences in proper- ties are being used (Mackaness, Auclair & Lagrange, 1973; Sher, Chaparas, Pearson & Chirigos, 1973) and (b) the viabilities of the preparations studied, as well as the experimental techniques used, vary from one laboratory to another. Lyophilized material can con- tain over 90% dead bacteria (Mackaness et al., 1973; Sher et al., 1973) and although dead BCG and their components can have immunological activity in their own right (Brown, Brown & 91jivi6, 1978; Kelly, 1976; Baker, Sharpton, Minden & Campbell, 1976), this is not necessarily the same as that of the intact and fully viable organisms. The work of Sultzer (1978) illus- trates the importance of these factors. He found that lyophilized BCG acted as a potent mitogen in vitro for splenic B cells but not splenic T cells. When used before lyophilizaton and injected i.v. into mice, how- ever, BCG organisms depressed the proliferation of 481
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Immunology 1979 38 481
Suppressed or enhanced antibody responses in vitro after BCG treatment of mice:importance of BCG viability
CAROLYN A. BROWN, I. N. BROWN* & V. S. SLJIVIC Departments ofImmunology and *Bacteriology,St Mary's Hospital Medical School, London
Acceptedfor publication 24 May 1979
Summary. Mycobacterium bovis, BCG, is known to becapable of either enhancing or suppressing variousimmune responses. Using a standard technique andnumber of organisms, some of the parameters pre-determining whether enhancement or suppression willoccur have been investigated. Dead BCG given intra-venously into mice caused an enhancement of theantibody response in vitro to sheep erythrocytes. Incontrast, the same number of viable organisms causedsuppression if given intravenously but enhancement ifgiven subcutaneously. The inclusion of 25% or morekilled organisms in an intravenous inoculum of fullyviable organisms changed suppression to enhance-ment. Treatment of BCG infected mice with strepto-mycin lessened the suppression but did not change it toenhancement. The possible causes of suppression arediscussed.
INTRODUCTION
Mycobacterium bovis BCG is an effective immuno-prophylactic for tuberculosis and is known to have arange of effects, from inhibition to potentiation, onvarious other immune responses. Attempts have
Correspondence: Dr C. A. Brown, Department ofImmunology, St Mary's Hospital Medical School, LondonW2 IPG.0019-2805/79/1100-0481$02.00C) 1979 Blackwell Scientific Publications
already been made to exploit its immunopotentiatingactivity for tumour immunotherapy (Baldwin &Pimm, 1978) and for the prevention of leprosy (Webb,Mims & Turk, 1979) but with equivocal results. Withfew exceptions, the assumption has been made thatBCG preparations which elicit an immunity effectiveagainst tubercle bacilli will also be those most effectivein the induction of anti-tumour and anti-leprosy im-munity. This may not be so. Although a wide literatureon the biological activities of BCG is developing, itsmode of action is still not fully understood. Compari-son of published results is difficult because (a) severalsubstrains of BCG with known differences in proper-ties are being used (Mackaness, Auclair & Lagrange,1973; Sher, Chaparas, Pearson & Chirigos, 1973) and(b) the viabilities of the preparations studied, as well asthe experimental techniques used, vary from onelaboratory to another. Lyophilized material can con-tain over 90% dead bacteria (Mackaness et al., 1973;Sher et al., 1973) and although dead BCG and theircomponents can have immunological activity in theirown right (Brown, Brown & 91jivi6, 1978; Kelly, 1976;Baker, Sharpton, Minden & Campbell, 1976), this isnot necessarily the same as that of the intact and fullyviable organisms. The work of Sultzer (1978) illus-trates the importance of these factors. He found thatlyophilized BCG acted as a potent mitogen in vitro forsplenic B cells but not splenic T cells. When usedbefore lyophilizaton and injected i.v. into mice, how-ever, BCG organisms depressed the proliferation of
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splenic B lymphocytes in the presence of PPD butenhanced that of lymph node B cells, with the Mon-treal substrain of BCG being more active in enhancingand suppressing than the Pasteur substrain. When thenon-lyophilized organisms were administered sub-cutaneously, however, they had no effect on the pro-liferation of the splenic B cells but still enhanced thatof the lymph node B lymphocytes.The object of the-present study was to define some of
the conditions in one test system which predeterminewhether suppression or enhancement of an immuneresponse will occur. The Glaxo strain of BCG wasused throughout since this is the strain readilyavailable in this country. The effect of its adminis-tration to mice on the capacity of their spleen cells togive primary antibody responses in vitro was followedduring the firstfew weeks after injection. The impor-tance of various parameters, especially the proportionof viable organisms in the inoculum, was assessed.
MATERIALS AND METHODS
MiceFemale CBA mice, usually 3-4 months old, bred in ouranimal house, were used throughout.
BCGThe Glaxo strain was used within 6 h of harvest from14 day suspension cultures in Glaxo glycerol-freemedium. The number of bacteria present in each sus-pension was estimated indirectly from its opacity froma curve, constructed at the beginning of the study, ofopacity against colony-forming units (CFU). Opacitywas measured using a Spekker (Hilger & Watts Ltd,London). Direct counts were made on some of thebacterial suspensions retrospectively by plating suit-able dilutions of culture in glycerol-free medium onMiddlebrook 7H10 agar plates (Difco) which werethen incubated at 370 for 21-28 days. Mice were in-jected i.v. via the tail vein or s.c. near the base ofthe tailusing bacterial suspensions diluted with glycerol-freemedium. Some ofthe BCG suspensions were heated at700 for 1 h; the heated bacteria produced no colonieson Middlebrook 7H10 agar plates and will be referredto below as 'heat-killed'.
The antibody response in vitroSpleen cell suspensions were prepared from mice(usually two from each group) at various time inter-vals after the injection of BCG. The medium used
throughout was RPMI 1640 containing added L-gluta-mine (Flow Laboratories, Irvine, Scotland), with0-024 M sodium bicarbonate, 5% foetal calf serum(Flow Laboratories) and 10 u/ml gentamicin. Thespleens were finely chopped with scissors before beinggently pushed and washed through stainless steelsieves. The cell suspensions were allowed to stand atroom temperature for 5 min and then decanted fromany cell clumps. The concentration of nucleated cellswas adjusted to 2 x 107 cells/ml before the cells wereincubated in modified Marbrook chambers (Mar-brook, 1967). One millilitre of cell suspension wasincubated in the inner tube of each Marbrook vesselwith either 2 x 106 sheep erythrocytes (SRBC) or 20 ngdinitrophenylated Ficoll (DNP-Ficoll). Disposableplastic universal containers (Sterilin) were used as theouter chambers of the Marbrook vessels and had 17-5ml tissue culture medium added to them. Thechambers were incubated at 370 in a humidified atmos-phere containing 5% CO2. The number of haemolyticplaque-forming cells (PFC) against SRBC or TNP-coated SRBC (Rittenberg & Pratt, 1969) was assessed4 days later by the method ofCunningham & Szenberg(1968).The results are recorded as arithmetic mean
numbers of PFC/culture from triplicate or quadrupli-cate cultures. In order to enable the plotting of resultsfrom different experiments in the same figure, the re-sponse of cells from BCG-treated mice is sometimesgiven as a percentage of the response of cells fromuntreated donors in that experiment. Responses ofcontrol cells varied from one experiment to another asseen from Tables 1-5.
Spleen sizesThe mean total number ofcells harvested from a groupof spleens was always recorded to give an assessmentof change in spleen size. The probable total number ofantibody-producing cells in the spleen was calculatedfrom the number of PFC against SRBC found in theMarbrook cultures (containing 2 x 107 cells) and thetotal number of cells in the spleen.
RESULTS
Effect of live or killed BCGThe antibody responses in vitro to SRBC of spleen cellstaken from mice at various times after i.v. injection of108 live or killed BCG are shown in Fig. 1. Spleen cellsfrom mice injected with live BCG (Fig. IA) gave an
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Antibody response in vitro after BCG treatment
enhanced PFC response to SRBC at 2-5 days, fol-lowed by a suppression which lasted until about 3weeks after infection. The initial enhancement was notaccompanied by an increase in the backgroundnumber ofPFC in cultures without antigen (results notshown).
In contrast, spleen cells from mice injected withheat-killed BCG (Fig. I B) gave a two- to six-fold
Figure 1. Effect of i.v. administration of 108 viable (A) orkilled (B) BCG organisms to mice on the antibody response invitro to SRBC. The number of PFC/culture of spleen cellsfrom treated mice is given as percentage of the response ofcells from untreated donors. Each symbol indicates a separ-ate experiment with a different batch of BCG organisms.Identical symbols occurring in both A and B indicate thesame batch of BCG. All values were obtained from triplicateor quadruplicate cultures of cells pooled from two or moremice.
enhanced anti-SRBC PFC response at some timebetween 12 and 27 days after priming. Some suppres-sion occurred, but this was confined to a few days afterthe enhancement peaks.The time of onset and the extent of suppression and
enhancement varied somewhat between experiments.Experiments described under the next two subhead-ings were carried out to investigate the possibility thatthis was due to batch variation either in the number orthe viability of BCG organisms.
Dose dependenceMice were injected i.v. with 107 or 108 organisms froma single batch of BCG and their spleen cells wereprepared and cultured on days 15, 36 or 57 afterinjection. The numbers of anti-SRBC PFC producedin vitro by spleen cells from mice injected with live ordead BCG at these concentrations are shown in Fig. 2.
ZQO
a)T
0E
Q)
a)
,5
a)
Days after BCGFigure 2. Effect of dose and viability of BCG administered tomice i.v. on the antibody response in vitro to SRBC. Thenumber of PFC/culture of spleen cells from treated mice isgiven as percentage of the response of cells from untreateddonors. Triangles, 107; circles. 108 organisms; open symbols,viable; filled symbols, killed BCG.
Administration of 108 live organisms resulted ingood suppression of the PFC response in vitro on days15 and 36. The suppression at 15 days caused by 107live organisms was less than that produced by 108 liveorganisms and had disappeared by day 36 after infec-tion. By day 57 the response produced by both doseshad recovered to above that of untreated mice.The onset of enhancement of the PFC response by
heat-killed BCG was also dependent on the number oforganisms administered. Fifteen days after injection,only the higher dose produced enhancement; by 36days 107organisms had produced enhancement. By 56days, both doses gave responses at near normal levels.
Effect of mixtures of live and killed BCGTo check whether the slight variations between experi-ments using live BCG could also be due to differingnumbers of non-viable organisms in the various BCGbatches, heat-treated organisms were mixed with fullyviable organisms of the same batch in known propor-tions. The mixtures of live and dead organisms werethen given i.v. so that all mice received a total of 108
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Carolyn A. Brown, I. N. Brown & V. S. Sljivic,
Table 1. Effect of various proportions ofviable and killed BCG organisms adminis-tered to mice on the response to SRBC invitro
Mice were injected i.v. with 108 BCGmade up of various proportions of viableand killed organisms and the response oftheir spleen cells was assayed 15 days later.
bacteria. For comparison, some mice were injectedwith 108 fully viable or 108 dead organisms. The PFCresponse of cells from all these mice was tested 15 dayslater (Table 1). Whereas 108 fully viable organismscaused suppression of the in vitro antibody response,the inclusion of 25% dead BCG in the inoculumresulted in a two-fold increase in the number of PFCagainst SRBC compared with the response from un-treated mice. The inclusion of 75% or more deadorganisms resulted in an even greater enhancementwith 108 organisms causing a four-fold increase overthe normal value.
Response to DNP-FicollThe effect of administration of live BCG on the anti-body response to the thymus-independent antigenDNP-Ficoll was investigated by culturing spleen cellstaken at intervals after the injection of 108 liveorganisms. Aliquots of the spleen cell suspensions pre-pared at each time interval from both infected andcontrol mice were also stimulated with SRBC for com-parison. Whereas suppression of the responses to bothantigens was marked in each of the five experimentscarried out, the relative recovery rates were found tovary. Representative results are given in Table 2.
Streptomycin in vivoThe results in Fig. I show that administration to miceof 108 viable rather than heat-killed BCG organismshad a marked suppressive effect on the anti-SRBC
Table 2. Effect of 108 viable BCG given i.v. on responses toSRBC and DNP-Ficoll in vitro
PFC/cultureDays after infection
SRBC DNP-Ficoll
9 Control 3427 + 58BCG 870+ 158
16 Control 3245 + 815 9955+ 1949BCG 135+43 925+ 114
30 Control 2395 + 182 5245+424BCG 1845+ 138 3680+250
PFC response in vitro of spleen cells 10-15 days aftertreatment. In order to see whether killing BCGorganisms in vivo could reverse the suppression, miceinjected i.v. with 108 live BCG were also given strepto-mycin sulphate (I mg intraperitoneally) on both thefourth and fifth days after infection. Uninfected micewere treated with streptomycin at the same times. Theresults are given in Table 3. Whereas streptomycin had
Table 3. Effect of treatment with streptomycin on the BCG-induced suppression of the antibody response
* Mice were infected i.v. with 108 viable BCG on day 0,received 1 mg streptomycin on days 4 and 5, and the PFCresponse against SRBC of their spleen cells was assayed attimes shown.
no effect at the times studied on the numbers of PFCfrom uninfected mice, it did lessen the suppressiveeffect of the BCG on anti-SRBC responses. There wasno indication of enhancement of the response. In afurther experiment (results not given), in which strep-tomycin was given i.p. immediately before intravenouslive BCG and again a week later, the suppression wasunaffected by 0-25 mg but abolished by 1 mg. Noenhancement was detectable by day 25 after BCGinjection. Viable organisms were recovered from themice in this experiment on day 18.
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Table 4. Effect of the route of administration of BCG on thePFC response to SRBC
* Mice were injected with 108 viable BCG organisms.
Route of administrationSeveral authors have reported that the effect of BCGdepends on the route of administration (Huchet &Florentin, 1976; Doft, Merchant, Johannessen, Cha-paras & Sher, 1976; Sultzer, 1978; Turcotte, Lafleur &Labreche, 1978). In order to find out whether thisfactor affected antibody responses in the present sys-tem, mice were injected either s.c. or i.v. with 108 liveBCG organisms from the same batch. Antibody re-sponses in vitro of cells from these mice were deter-mined 10 and 17 days after injection and the results aregiven in Table 4. The PFC values after BCG adminis-
tered i.v. showed the usual suppression at day 10followed by recovery at day 17 but the same suspen-
sion of organisms given s.c. caused marked en-hance-ment of PFC at day 10 followed by approximatelynormal values at day 17. No attempt was made tofollow the full time course of the effect of BCGadministered s.c. Further results, including the effectof the route of administration on spleen size (seebelow) are given in Table 5. Preliminary experimentsindicated that dead BCG given s.c. had little effect onin vitro responses to SRBC at least at the time intervalsstudied.
Spleen size and method of expressing PFC results
The greatest increase in cell yield was obtained afterinjection of live BCG i.v. (Table 5). Although thespleens from mice injected i.v. 13 days previously withlive BCG contained two to three times the number ofcells as the spleens from untreated mice, the number ofPFC/spleen was still less than half that of the normalvalue. On day 17, however, when the number ofPFC/culture was the same as that for untreated mice,
Table 5. Comparison between results for numbers of cells producingantibody in vitro against SRBC expressed as PFC/unit number ofspleen cells and as PFC/total spleen
* 108 live (L) or dead (D) BCG were injected i.v. or s.c.t Time after BCG injection before removal of spleens.t Number of ml containing 2 x 107 nucleated cells (average yield
from at least two mice).§ Arithmetic mean+ SEM of PFC against SRBC. The figures in
parentheses are the results expressed as percentages of the response ofuntreated mice.
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because the cell yield was more than four times thevalue for normal mice, the PFC/spleen was corres-pondingly higher. Similarly, by day 20, although thePFC/culture was the same as that for untreated mice,the PFC/spleen was five times higher. Dead BCGgiven i.v. also increased the cell yield and thereforePFC/spleen but to a lesser extent. BCG (live or dead)administered s.c. had little effect on spleen cell yields atthe times studied.
DISCUSSION
The course of systemic BCG infection in the mouse hasbeen described (Sher et al., 1973; Bekierkunst, Levij,Yarkoni, Vilkas, Adams & Lederer, 1969). Typically,in our mice, the numbers of bacilli in the liver andspleen increase over the first 2 weeks of infection andthen begin to fall. Multiplication of bacilli is asso-ciated with both liver and spleen enlargement whichpersists for some time after the infection has beencontrolled.The initial enhancement of in vitro PFC responses
observed within the first week of infection with liveBCG could be due to soluble products ofBCG (Mack-aness et al., 1973) or to polyclonal B-cell stimulation,which has been found to peak in the spleen 3 days afterBCG injection but to subside by the fourth day(Sultzer, 1978). No indication of polyclonal B-cell sti-mulation was found in our system, however. The slightvariation in onset and extent of suppression and en-hancement are presumably due to differences in thenumber of organisms injected since a deliberate ten-fold alteration in number caused a marked differencein response. BCG grows slowly on solid media andconventional assessment of the number of viableorganisms by colony count was thus not appropriatehere. We therefore standardized BCG suspensions bydetermining their opacity and comparing this with apreviously determined standard curve of opacityagainst colony-forming units. We appreciate that opa-city measurements might not detect small differencesin total count. Even allowing for these slight varia-tions, the main effects occurred within 2-3 weeks afterBCG injection. This time is known from other studiesto be the period when BCG stimulates host activityagainst tumours (Kelly, 1976), bacteria (Blanden, Lef-ford & Mackaness, 1969) and blood parasites (Clark,Wills, Richmond & Allison, 1977).Many bacterial infections, including BCG, cause
architectural disturbances in tissue which can
markedly affect immune responses in vivo e.g. byaffecting the distribution of systemically or parenter-ally administered antigen. In vitro assays of immunecapacity, such as the one used here, are useful in suchsituations because they measure the response of astandard number of cells to a standard antigenicstimulus. They are also invaluable for the assessmentof the role of different cell types in altered responses byusing fractionated and enriched cell populations. Thiswill be the subject of a later communication on effectsof BCG. There are, however, certain aspects of inter-pretation of the present results that need to bediscussed. In the experiments we have described thetreatment causing maximal increase in spleen size, i.e.live BCG given i.v., also caused maximal suppressionof the response in vitro to SRBC and DNP-Ficoll.Optimal responses to SRBC in culture depend on thepresence of the correct number not only of B cells butalso ofT cells and macrophages; the response to DNP-Ficoll is T-cell independent. Since responses to bothantigens were depressed this could be due to the dilut-ing out of functional B cells caused by increasednumbers of other cell types, including haematopoieticcells in the mouse spleen. This is not an adequateexplanation, however, because maximal suppressionoccurred before maximal spleen size was reached andthe PFC counts per culture returned to normal levelswhile the spleen was still much enlarged (cf. Table 5).A further consequence ofa change in spleen size is thatthe in vitro response obtained with a standard numberof cells may no longer adequately reflect the antibody-producing potential of the whole spleen. To allow forspleen size alteration, the number of PFC/spleen canbe calculated from the mean value obtained from theculture chambers and the total number of nucleatedcells in the spleen. Any extrapolation of this kind,however, should be made cautiously.BCG is a widely used adjuvant and is known to
affect the various cell types involved in antibodyproduction, stimulating both macrophages (Bruley-Rosset, Florentin, Khalil & Mathe, 1976) and lympho-cytes (Bekierkunst, 1976). Cell co-operation isprobably required for stimulation of either cell type tooccur (Mokyr & Mitchell, 1975). Reports on the effectof BCG on antibody responses in vivo are variable.Administration of live BCG (106 Phipps) into the foot-pad of mice increased numbers of PFC in the draininglymph node against SRBC given by the same route(Miller, Mackaness & Lagrange, 1973). In a detailedstudy of various effects of live Pasteur BCG ('less than7 days old', 7 x 106 viable units/mouse), Florentin,
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Antibody response in vitro after BCG treatment 487
Huchet, Bruley-Rosset, Halle-Pannenko & Mathe(1976) reported that s.c. administration had littleeffect, whereas i.v. administration markedly enhancedsplenic PFC responses in vivo to three differentantigens given 14 days after BCG. In contrast, Doft etal. (1976) found suppressed splenic PFC responses toboth thymus-dependent and independent antigensgiven in vivo 12 days after an i.v. injection of PhippsBCG (106 CFU from batches frozen at -70°). In viewof the results presented here on the different effects oflive and dead BCG and the importance oftheir relativeproportions in an inoculum, we suggest that some ofthe apparent contradictions concerning the effect ofBCG on spleen cells and antibody production (andpossibly effects on tumours?) can be attributed todifferences in the proportion of viable organisms ininocula in addition to differences between BCG strainsused.
Responses other than antibody production can alsobe suppressed by BCG injected i.v. Thus, spleen cellsfrom treated mice showed a marked reduction of re-sponsiveness to T-cell mitogens PHA and Con A(Huchet & Florentin, 1976; gljivic, Brown & Brown,unpublished results). In addition, such cells sup-pressed mixed lymphocyte and graft-versus-host reac-tions (Gefford & Orbach-Arbouys, 1976) and[3H]-thymidine uptake by both PHA-stimulated lym-phocytes and tumour cells (Orbach-Arbouys & Pou-pon, 1978), suggesting that suppression of an immuneresponse by agents such as BCG does not necessarilypreclude their use for cancer immunotherapy. Moreinvestigation is needed on this aspect, however.The administration of streptomycin to mice either
immediately before live BCG i.v. or 4 and 5 days laterlessened the suppressive effect considerably but didnot convert it to obvious enhancement, at least at thetimes studied. The antibiotic must have been acting asa bacteriostatic rather than as a bactericidal agentsince other experiments showed that the presence of25% or more killed organisms in an inoculum causedenhancement. Moreover, viable organisms weredetectable by culture 18 days after BCG and strepto-mycin administration which would imply that activemultiplication rather than maintenance of viability isresponsible for suppression.
Further investigation is needed to elucidate fully themechanism of the suppression induced by viable BCGgiven i.v. Presumably suppression reflects either thefailure of a particular cell type to function properly orthe production of suppressor cells or factors. In vitroresponses to SRBC require the co-operation ofmacro-
phages, B cells and T cells and the failure to respondfully could be due to some adverse effect on one ormore of these cell types. Since responses to DNP-Ficoll, a T-cell independent antigen, were also sup-pressed a direct action on T cells alone seems unlikely.Some BCG organisms would be metabolizing andpossibly multiplying within macrophages in the cul-ture chambers (viable organisms are present, unpub-lished observation) particularly in the early stages ofinfection and could affect the normal functioning ofmacrophages, as suggested for Taenia crassiceps(Miller, Good & Mishell, 1978) and malaria (Brown,Watson & Sljivi5, 1977) infections in mice. Since, how-ever, responses to DNP-Ficoll are relatively macro-phage independent and, in the five experiments wehave reported, responses to both DNP-Ficoll andSRBC were suppressed, a defect of macrophage func-tion seems unlikely as the sole explanation. Anotherpossibility is that there was active suppression by cellsor their products of the cells involved in antibodyproduction. This is supported by the reports of sup-pressor T cells (Sultzer, 1978), possible suppressormacrophages (Doft et al., 1976) and suppressor adher-ent cells inhibiting T-cell function (Florentin et al.,1976). Huchet & Florentin (1976) suggest that thenature of the suppressor cell is dependent on the BCGdose; they found suppressor adherent cells and, at ahigher dose, suppressor T cells in addition.We have not tested directly the relevance of our
findings to tumour therapy but two recent publica-tions (Griffith & Regamey 1978; Baldwin & Pimm,1978) stress the need for basic research on the biologyof BCG in connection with its effects on immuneresponses. Our study shows that the viability ofa BCGpreparation is ofparamount importance in this type ofexperiment and grossly affects the type of responseelicited. By inference, similar considerations couldapply to BCG preparations used for tumour therapy.
ACKNOWLEDGMENTS
This investigation was supported by the CancerResearch Campaign. We thank Gwen Dewey for tech-nical assistance and Glaxo Laboratories Ltd, Green-ford for the BCG suspensions.
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