Imminology 1978 34 807
Further studies on the H-2 linked dependence of the adjuvant action of Brucella abortus
D. 0TH & D. SABO LOVI C INSERM U.95 Research Unit, Plateau de Brabois, 54500 Vandoeuvre, France
Received 12 May 1977; acceptedfor publication 26 September 1977
Summary. The B 19S strain of Brucella abortus is found to act as an adjuvant to the anti-sheep red blood cell (SRBC) reaction in some congenic strains of mice but not in others. If the recipient is H-2b, there is no adjuvant effect (B 19S); neither is there (thymus-dependent) anti-B 19S reaction as measured by thymocyte activation and change of electrophoretic mobility. In contrast, there is a thymus-independent anti-B 19S reaction (production of haemagglutinins) as good in the H-2b mice as in the others. Experiments with T cell deprived mice show that the adjuvant action of B 19S is thymus-dependent. As the antiSRBC reaction without adjuvant is also thymusdependent, it is difficult to distinguish anti-SRBC and anti-B 19S reactions from the adjuvant action of B 19S on the anti-SRBC reaction. Several explanations are possible, all involving H-2 (Ir?) controlled thymus dependent mechanisms.
It was found that the A/Sn and A.CA strains displayed a strong sensitivity to the well characterized adjuvant action of Brucella (Toujas, 1975), while the A.BY did not. This strain was also found to be very different from the others with regards to the kinetics of splenomegaly following an injection of this adjuvant (Oth, Sabolovic & Le Garrec, 1974). The action of many adjuvants has been shown to be thymus dependent (Allison & Davies, 1971; Del Guercio, 1972). As the genetic difference existing between the presently studied strains lies at the level of the H-2 complex, which contains thymus controlled immune response (Ir) genes (Benacerraf & Katz, 1975), both confirmation of H-2 influence and thymus dependence of the observed adjuvant action were the aims of these studies.
MATERIALS AND METHODS Animals The four congenic resistant strains A/Sn, A.CA, A.SW, A.BY were donated by Dr Eva Klein, Karolinska Institutet, Stockholm. Another congenic resistant pair was used, B 10 and B 10.A. This was a gift of Drs M. and J. Colombani, Hopital St Louis, Paris. The animals were maintained by strict brother x sister mating in our colony, under conventional conditions, in temperature controlled facilities. They were used when 4-8 months old. For a given experiment, they were age, weight and sex matched.
INTRODUCTION We have previously shown a differential adjuvant action of Brucella abortus, as measured by the increase of the number of splenic cells able to form haemolysis 'plaques' on sheep red blood cell layers, in three congenic strains which differ by the H-2 genetic complex (Sabolovic, 0th & Burg, 1971).
Correspondence: Dr Daniel 0th, INSERM U.95 Research Unit, Plateau de Brabois, 54500 Vandoeuvre, France. 0019-2805/78/0500-0807 $02.00 (D) 1978 Blackwell Scientific Publications B
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Antigens Sheep red blood cells (SRBC) were purchased from the Institut Pasteur Production, Paris. They were washed three times and used as a 10% dilution in saline. Brucella abortus, strain 19 in smooth colonies (B 19S), was heat inactivated (1 h, 650). This was kindly offered by Dr Y. Le Garrec, Service du Professeur Pilet, Ecole Nationale Vt6rinaire, MaisonsAlfort. It was injected as a suspension in sterile saline.
Typing of H-2 haplotype Some (A.CA x A.BY)F2 hybrids were typed for their H-2 haplotype with antisera prepared by multiple intraperitoneal injections of A.CA splenic cells to A.BY and vice versa. The test employed was the haemagglutination technique as described by Kaliss (1973). Plaque forming cells The number of direct plaque forming cells (PFC) was determined exactly as previously (Oth et al., 1974; Sabolovic et al., 1971). Usually, five mice were used for each determination.
Activation of thymus cells Thymus cells were injected intravenously into ten recipients freshly. irradiated with 900 rad X-rays (180 kV, 1 mm Cu filtration) which had been injected 30 min intraperitoneally with 100 i.u. heparin before. Shortly after these treatments, some of the mice received B 19S intraperitoneally, while others didnot. Among B 19S injectedmice, some diedduring the following hours. After 3-5 or 4 days, the mice received intraperitoneally 25 pCi oftritiated thymidine (3H-TdR), purchased from Commissariat a l'Energie Atomique, Gif-sur-Yvette, and with a specific activity of 26-27 Ci/mM. Half an hour after this injection, they were killed, their spleens were removed and dessicated. Radioactivity determinations of the spleens
performed by burning the spleens in a TriCarb 305 sample oxidizer (Packard, Downers Grove, Illinois) and counted in a liquid scintillation counter (type SL30, Intertechnique, Plaisir). The ratio: mean splenic radioactivity of mice injected with B 19S mean splenic radioactivity of non-injected mice was determined for each group.
were
Electrophoretic mobility (E.M.) determination This is determined in the conditions already described (Wioland, Sabolovic & Burg, 1972), using a cylindrical microelectrophoresis apparatus (Mark I, Rank Bros, Bottisham, England). The values are expressed as jpm s-I V-1 cm. Preparation of T lymphocyte 'deprived' mice The procedure described by Davies, Leuchars, Wallis & Koller (1966) was used. 2-month-old mice were thymectomized. 2 months later, they were heavily irradiated with X-rays (850rad, with 1 mm Cu added filtration) and intravenously injected with 5x 106 marrow cells. After a further 3-4 months they were ready to use. Titration of anti-Brucella agglutinins The sera of three to six mice per group were pooled and two-fold diluted in saline, starting from 1/10. Several batches of Brucella test antigens were purchased from Institut Pourquier, Montpellier. Complete agglutination, according to Wright's method, was read after 24 h at 370, in a 1 ml total volume.
RESULTS Confirmation of the dependence on H-2 linked genes of the adjuvant action of B 19S on plaque forming cells It was previously shown (Oth et al., 1974; Sabolovic
Table 1. F2 analysis of the action of the H-2 complex on the adjuvant effect of Brucella the number of SRBC-PFC. (A.CA x A.BY)F2 hybrids are H-2 typed according to haemagglutination test on
H-2 type*
Brucella treatment
H-2t/H-2'
No Yes No Yes
H-2rIH-2r H-2b/H-2b
H-2b/H-2b
PFC per spleen + SE PFC per 106 cells + SE
26,250± 5625t 104,000 ± 20,954 25,468 ±9952 27,812 ± 5
38 0 ±12-5 73-3 ± 11-9 40-1 ±14-6 22-8 ± 5-3
H-2(/H-2b hybrids were used for another purpose. t Mean of two to five mice. *
H-2 linked dependence of adjuvant action of Brucella abortus
809
Table 2. Test of adjuvant effect of Brucella on PFC in BlO.A and BlO mouse strains
Strain
H-2 type
BlO.A
H-2a
B1O
Brucella treatment PFC per spleen ± SE PFC per 106 cells ± SE No Yes No Yes
H-2b
*
64,874 ±28,026*
448,249 + 118,978 54,749 +24,486 103,437 +25,110
252 6 ± 101-5 1220-8 ±676-1 198-4 ± 100-6 184-4 ±45-3
Five mice per group.
al., 1971) that an intravenous injection of 500600 ug of killed B 19S, given 30-32 days before the PFC assay, results in a large increase of PFC numbers in the A/Sn (H-2a) and the A.CA (H-2t) strains, while this is either modest or non-existent in the A.BY (H-2b) strain. To check further the role of the H-2 complex in this difference, we used (A.CA x A.BY)F2 hybrids which were typed by haemagglutination for their H-2 haplotype. They received intravenously 600 pg of B 19S, 27 days before the PFC assay. As seen from the results in Table 1, the A.CA-like H-2f/H-2f mice developed a -4 fold increase in the PFC number, while the A.BY-like H-2 /bH-2b did not demonstrate any increase. The H-2fIH-2b heterozygotes have been used for another experiment and have not been tested here. Previous studies showed that they behave like the A.CA parental strain (Oth et al., 1974). That the H-2a haplotype gives a better adjuvant action of B 19S than the H-2b haplotype was also verified in another genetic background. Mice of the B 10 (H-2b) and B 10.A (H-2a) strains were injected et
with 500 pg B 19S, 34 days before PFC assay. As seen from Table 2, the increase of PFC per spleen was much more pronounced in the H-2' than in the H-26 mice, where no increase of PFC per 106 nucleated cells (spleen cells) was (again) observed. So both these results strongly argue in favour of the importance of the H-2 haplotypefor the presently studied adjuvant action.
Comparison of in vivo thymus cell activation by B 19S, in the A/Sn A.CA, A.SW and A.BY strains Although the Brucella antigens have been considered as thymus-independent antigens (Takahashi, Mond, Carswell & Thorbecke, 1971) certain results suggest some thymus dependence of the maturation of cells sensitive to high levels of these antigens (Gras, Morros, Guix & Tuset, 1975) and a strong activation of thymus cells has been demonstrated (Krueger & Gershon, 1972). We therefore injected intravenously 3-5 x 107 syngeneic thymus cells into lethally irradiated recipients. Within 1 h after irradiation,
Table 3. Activation by B 19S of thymus cells injected in lethally irradiated mice
Strain (H-2 haplotype)
No. of injected thymocytes
Amount of B 19S injected after irradiation (mg)
A/Sn (a)
5 x 107 3 x107 5 x 107
2-5t
A.CA (f)
3x107 A.SW (s)
5x 107
3x107 A.BY (b)
5 x 107
3xl10 * Separating
1-5 2-5 15 2-5 15 25 1-5
Time interval* (days)
3.5 4-0
3-5 40 3-5 4-0 3-5 4-0
irradiation and killing.
t Spleen radioactivity of a 19S injected mice Spleen radioactivity of non-injected mice
t Mortality before 3.5 days was frequently observed with this dose.
Ratiot
4.3, 4-4 2-3 4-1, 6-9 3-1 5-2 6-2 0-8, 2-9,
1-3
1*0
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D. 0th & D. Sabolovic
some of the treated mice received intraperitoneally 1 5-2 5 mg of the B 19S preparation, while some other remained uninjected. After 3 -5-4 0 days, all the mice received an injection of 3H-TdR, and the ratio: spleen radioactivity of Brucella injected mice spleen radioactivity of non-injected mice was determined. Several experiments were performed, using three to five mice per group each time. The results in Table 3 show that this ratio is always lower in the A.BY mice than in the three other strains, in different experimental conditions. This would suggest that the thymus cell activation by Brucella, as defined by Krueger et al. (1972) is lower in theformer strain than in the three others.
moving B lymphocytes and fast moving T lymphocytes (Wioland et al., 1972). In the case of nontreated mice belonging to the strains A/Sn, A.CA and A.BY, the values of E.M. of both B and T lymphocytes are very similar, varying from 0-68+ 0 01 to 0-69±0-01 for the B cells, and from 1-14+ 0 01 to 1-16±0-01 for the T cells (Donner & Wioland, 1976). If the B 19S is injected a few days before, one observes a dramatic change in the E.M. of A/Sn or A.CA spleen cells, characterized by a diminution of the E.M. of the T cells and an aug-
Differential effects of B 19S on the electrophoretic mobility of spleen cells of A.BY and the other strains Electrophoretic mobility determination of lymphocytes permits a very accurate distinction of slow T
B
A.BY
151
1-
0
z
U
0
z
T
B
A/Sn 15_ I
4
I
10 5
nl
1.5 i-0 0-5 E.M. (Am s"' VW'cm) Figure 1. Histogram of E.M. (number of cells as a function of electrophoretic mobility value of each individual cell) of the lymphoid spleen cells of A.BY (upper) and A/Sn (lower) mice, 8 days after intravenous injection of 200 pg of B 19S. The arrows indicate the mean values for B and T cells of
non-treated mice.
E.M. (pLm s-1 V'1 cm) Figure 2. Histogram of E.M. of spleen cells of A.BY (dotted line) compared with splenic cells of A/Sn (upper), A.CA (middle) and A.SW (lower) (continuous lines) 10 days after i.v. injection of 600,ug of B 19S. The arrows indicate the mean values for B and T cells of non-treated mice.
H-2 linked dependence of adjuvant action of Brucella abortus mentation of that of B cells. Several experiments of that sort have been performed, also using the Peyer patches lymphoid cells as a test for the sensitivity to B 19S injection (to be published). In this regard, the A.BY strain is much less sensitive than the other tested strains. A typical experiment is shown in Fig. 1, where the E.M. pattern of spleen cells of A/Sn and A.BY have been determined 8 days after an intravenous injection of 200 ug of B 19S. As the T cells E.M. of the A/Sn is reduced to 1 -05 ± 0 06, that of the A.BY remains almost unchanged, with a value of 1-13±0-07. Although the E.M. of the A/Sn B cells is augmented to the value of 0-86+0-079 that of the A.BY is less so, with a value of 0-75 ±0 1. Similar results have been obtained comparing A/Sn and A.BY mice, 10 days after 200 ug of B 19S (not shown). Using a larger amount of B 19S, e.g. 600 pg and measuring the E.M. after 10 days, the diminution of the E.M. value of the T cells is more dramatic, except for the strain A.BY. Fig. 2 shows such an experiment, comparing A/Sn, A.CA, A.SW and A.BY strains. However, with such an amount of
811
B 19S, the reaction seems to involve both B and T cell populations with more intensity than with 200 pg. One observes fluctuations in the cell E.M. distribution also in the A.BY strain, but the slowing of the T cells (right on the histogram) is always more pronounced in A/Sn, A.CA or A.SW (continuous line) than in the A.BY (dotted line). One observes a relative quantitative diminution of the slow B cell population after B 19S injection. This is in agreement with the observation that the proportion of immunoglobulin positive lymphocytes (mostly B cells) diminishes after such injections (Dumont, personal communication). Thus, using this criterion, the A.BY seems also to react less in vivo to Brucella antigens than to the A/Sn or A.CA and A.SW, particularly at the level of the T cells. This difference seems rather qualitative than quantitative, because the number of T cells in the A.BY is of the same order of magnitude as in the other strains: data in Tables 1 and 4 permit one to calculate that the number of nucleated spleen cells is the same in the four strains, after an injection of B 19S, and the A.BY has the same B/T
Table 4. Test of adjuvant effect of Brucella on PFC in T cell deprived and control mice of the four congenic resistant strains
Strain A/Sn
Status of mice
Brucella treatment*
Sham
No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes
Deprived A.CA
Sham
Deprived A.SW
Not treated:
Sham
Deprived A.BY
Sham
Deprived
PFC per spleent ± SE
61,249 + 37,920 212,812 ± 16,875 833 ±551 312±312 12,604 + 6988 11,718 +2656 1354 ± 375 1875 4218 ±3906 43,906 ±4844 8734 ±4377 17,812 1406 ±469 2187 7187 ±5013 3624 ±794 1562 ± 1097 937±127
PFC per 106 cells ± SE 222 ± 160 330 ±43 1-7 ±0-8 0-2±0-2 25 ± 13 10 ±0 4 6 ± 1-8 2-8 14-8 ± 14-1 50 3 ± 10-5 30 ± 19 47 4-6 ±2 7-9 12 5 4 1 39±I
2-7±09
500 pg of B 19S injected intravenously 26 days before the PFC determination. to five mice, except where individual value given (one mouse). $ This group was added to confirm that Brucella has an adjuvant effect on PFC in normal mice, as previously found with A/Sn and A.CA. Their age is 6 months.
t Two
D. 0th & D. Sabolovic
812
cell distribution as the A/Sn and A.CA (Donner & Wioland, 1975, 1976). Thymus dependence of the adjuvant effect of B 19S on PFC The previous results suggest that the A.BY strain does not respond to the B 19S adjuvant action because of an H-2 linked defect of thymus sensitivity to these antigens. This was checked by comparing the adjuvant action in 8-month-old male mice belonging to the four congenic strains, in the presence and absence of T lymphocytes. T cell 'deprived' mice were obtained according to Davies et al. (1966) (see Material and Methods) and compared with sham-operated, T cell containing partners. Normal, non-operated A.SW male mice were also responsive to the adjuvant effect of B 19S, as this was previously shown to be the case with A/Sn and A.CA mice (Sabolovic et al., 1971). The results in Table 4 show that the T cell deprived mice of the four strains behave approximately in the same way: not only is the number of PFC dramatically diminished in the deprived mice, but the adjuvant effect of B 19S is completely abolished in all cases. Among the sham-operated (control) mice, the absence of adjuvant effect found in the case of A.BY
is expressed by not only an absence of augmentation of PFC, but by a decrease of this number. In the case of the A.CA, there is no change of the PFC number after B 19S, contrary to what happened previously with younger, non-treated mice (Oth et al., 1974; Sabolovic et al., 1971). On the other hand, sham-operated A/Sn and A.SW strains displayed sensitivity to the adjuvant effect of B 19S. This effect seems nevertheless much more expressed in the non-treated than in the sham-operated mice, as seen from the data obtained with A.SW. Table 4.
Comparison of anti Brucella agglutinins in the four congenic strains Production of anti Brucella agglutinins is a thymus independent event (Takahashi et al., 1971). We therefore wished to measure the total anti-B 19S agglutinins in the four congenic resistant strains. This was performed in age and sex matched mice, using different batches of Brucella test antigens (Table 5), as a function of the time after a single injection, or as a function of the amount of B 19S of one injection, after 32 days (Table 6). The results show that, in this case, the A.BY strain is certainly not a weaker reacter than the three others. On the contrary, it is often the most, or among the most, reactive strains,
Table 5. Reciprocal of the anti Brucella agglutinin titre as a function of the time following an intravenous injection of 500 pg of B 19S
Strain
Day 6
Day 12
Day 23
Day 40
Day 51
Day 75
Day 100
A/Sn A.CA A.SW A.BY
40 40 40 80*
160 160 160 320*
320 320 160 640*
320* 320* 80 320*
320 320 80 640*
40 20 80* 80*
0 20 0 40*
*
Highest value, or values, of the group.
Table 6. Reciprocal of the anti Brucella agglutinin titre 32 days after tion of B 19S, as a function of the amount of B 19S injected
Strain
A/Sn A.CA A.SW A.BY
an
intravenous injec-
250 pg
500 pg
850 pg
1000 pg
1250 pg
1500 pg
1750 pg
80 80 40* 160
160 160 80* 160
640 640 320* 640
640 640 640 640
1280 1280 320* 1280
320 160 80* 320
1280 1280 640* 1280
*
Lowest value of the group.
H-2 linked dependence of adjuvant action of Brucella abortus giving the earliest production of agglutinins and persisting for the longest time. Curiously, the A.SW strain, which has quite normal thymus dependent reactions, often displays a weaker production of agglutinins. This could be correlated with their relative poverty in B lymphocytes, as compared with the other strains (Donner & Wioland, 1975, 1976). The results suggest that the A.BY strain's defective reaction to B 19S is only restricted at the level of the thymus dependent events, but not at the level ofthe thymus independent reactions.
DISCUSSION The marked genetic influence of the genome, on the sensitivity of the adjuvant effect of B 19S on anti SRBC reaction, seems to be closely associated with the H-2 haplotype of the tested mice. This is confirmed in the present work with congenic mouse strains having two different genetic backgrounds, and also with H-2 typed F2 hybrids. Thus, the action of a non H-2 part of the genome on this phenomenon seems improbable. Genetic considerations suggest that the I region of the H-2 complex would be implicated, with a possible complementation effect (Melchers & Rajewsky, 1975) between the I-B and I-C subregion associated genes (unpublished observation). It is therefore tempting to attribute the H-2 influenced adjuvant effect of B 19S on anti SRBC reaction to Ir genes, which have been shown to control anti SRBC reactivity also in the absence of B 19S (Sabolovic et al., 1971). The question is now: wha tare the basic mechanisms of such a control? It must be considered that B 19S, as an adjuvant, is also strongly antigenic by itself. Also, both B 19S, as well as SRBC, are not simple antigens (Le Garrec, 1973). The Ir control of these several antigens could be exerted by several Ir genes, and the overall reaction observed (i.e. adjuvanticity, or absence of adjuvanticity) could result from interactions between these antigens and different Ir controlled reactions. Absence of an adjuvant effect observed with the H-2b haplotype could result from several mechanisms, including non-reactivity, antigenic competition, development of suppressor cells. The H-2b haplotype bearing mouse could be a 'poor responder' to those antigens present in B 19S, which are thymus dependent and able to stimulate an anti SRBC reaction non-
813
specifically, for instance by eliciting the synthesis of PFC stimulating factors (Mond, Takahashi & Thorbecke, 1972). On the other hand, as the H-2b mice are also poor responders to SRBC (Sabolovic et al., 1971) the observed results could be the consequence of a lower thymus-dependent reactivity of H-2b towards SRBC, and not necessarily of their observed poor thymus-dependent reaction against B 19S. With the present results, it is not possible to distinguish clearly if the observed facts result from the reactivity towards the tested antigen SRBC alone, or towards the adjuvant B 19S. On the other hand, the H-2b bearing animals were found to be quite reactive against the B 19S antigens when a thymus-independent reaction was studied. They also might be reactive to thymus-dependent antigens of B 19S, but in such a way that this reaction would result in either suppressive cells (which would block both anti B 19S and SRBC reactions), or in antigenic competition between different antigens contained in B 19S, or in both B 19S and SRBC (Toujas, Dazord & Guelfi, 1974). So, the balance between competition of potentiation of immune reactions against several antigens could be H-2 dependent, explaining the present results. Analysis of these individual reactions should be made, testing as 'adjuvant' different purified antigens of the B 19S preparation. A better characterization of the genetic dependence of the adjuvanticity of a B 19S preparation could be of practical use, because this adjuvant may exert some therapeutic or prophylactic action against some tumours, in some (but not all) strains of mice (Le Garrec, Sabolovic, Toujas, Dazord, Guelfi & Pilet, 1974).
ACKNOWLEDGMENTS We wish to thank Dr Yvonne Le Garrec and Dr Louis Toujas for continued interest, suggestions and discussion of the paper, and Mrs Marie Chantal Beugnot, Mr Pascal Mouchette and Mr Andre Liegey for technical assistance. The work was supported by the INSERM Paris, contract ATP 11-7432. REFEREN CES ALLISON A.C. & DAVIES A.J.S. (1971) Requirements of
thymus dependent lymphocytes for potentiation by adjuvants of antibody formation. Nature (Lond.), 233, 330.
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BENACERRAF B. & KATZ D.H. (1975) The histocompatibilitylinked immune response genes. Advanc. Cancer Res. 21, 21. DAVIEs A.J.S., LEUCHARS E., WALLIS V. & KOLLER P.C. (1966) The mitotic response of thymus-derived cells to antigenic stimulus. Transplantation, 4, 438. DEL GUERCIO P. (1972) Effect of adjuvants on the antibody response to a hapten on a thymus independent carrier. Nature (New Biol.), 238, 213. DONNER M. & WIOLAND M. (1975) Relationship between H-2 region and surface characteristics of the mouse spleen cells. Folia biol. (Prague), 21, 146. DONNER M. & WIOLAND M. (1976) Possible involvement of H-2 region in distribution control of T and B lymphocytes in spleen and in surface characteristics of T lymphocytes. Folia biol. (Prague), 22, 51. GRAS J., MORROS J.M., Guix J. & TUSET N. (1975) Stimulation of IgG antibody formation by sublethal irradiation during persistently repeated immunization with Brucella abortus. Immunology, 28, 629. KALISS M. (1973) An improved polyvinylpyrrolidone (PVP) method for titering mouse allohemagglutinins. Transplantation, 15, 251. KRUEGER J. & GERSHON R.K. (1972) DNA synthetic response of thymocytes to a variety of antigens. J. Immunol. 108, 581. LE GARREC Y. (1973) Variation lisse-rugueuse chez Brucella abortus: quelques aspects immunologiques et immunochimiques. These d'Etat Pharmacie, Universit6 de ParisSud. LE GARREC Y., SABOLOVIc D., ToUJAs L., DAZORD L., GUELFI J. & PILET C. (1974) Activity of inactivated
Brucella on murine tumors: prophylactic effect and combination with specific immuno-stimulation. Biomedicine, 21, 40. MELCHERS I. & RAJEWSKY K. (1975) Specific control of responsiveness by two complementary Ir loci in the H-2 complex. Europ. J. Immunol. 5,753. MOND J.J., TAKAHASHI T. & THORBECKE G.J. (1972) Thymusderived cell (T cell) activation by heterologous antigens as a replacement of specific immune T cells in the transfer of the secondary response to sheep erythrocytes. J. exp. Med. 136, 715. OTH D., SABOLOVIc D. & LE GARREC Y. (1974) Genetic (H-2) dependence of the duration of splenomegaly induced by Brucella abortus. Folia bio. (Prague), 20, 20. SABOLOVIC D., OTH D. & BURG D. (1971) Immunological reactivity to sheep red blood cells in three congenic resistant strains of mice. Immunology, 20, 341. TAKAHASHI T., MOND J.J., CARSWELL E.A. & THORBECKE G.J. (1971) The importance of 6 and Ig bearing cells in the immune response to various antigens. J. Immunol. 107, 1520. ToUJAs L. (1975) Les diftfrents m6canismes de stimulation des r6ponses immunitaires par des pr6parations inactiv6es de Brucella abortus. These de Doctorat Biologie Humaine, Universit6 de Paris-Sud. ToUJAs L., DAZORD L. & GUELFI J. (1974) Increase of Brucella induced immunostimulation by administration in combination with a specific antiserum. Rec. Results Cancer Res. 47, 302. WIOLAND M., SABOLOVIC D. & BURG C. (1972) Electrophoretic mobilities of T and B cells. Nature (New Biol.), 237, 274.
Further studies on the H-2 linked dependence of the adjuvant action of Brucella abortus
D. 0TH & D. SABO LOVI C INSERM U.95 Research Unit, Plateau de Brabois, 54500 Vandoeuvre, France
Received 12 May 1977; acceptedfor publication 26 September 1977
Summary. The B 19S strain of Brucella abortus is found to act as an adjuvant to the anti-sheep red blood cell (SRBC) reaction in some congenic strains of mice but not in others. If the recipient is H-2b, there is no adjuvant effect (B 19S); neither is there (thymus-dependent) anti-B 19S reaction as measured by thymocyte activation and change of electrophoretic mobility. In contrast, there is a thymus-independent anti-B 19S reaction (production of haemagglutinins) as good in the H-2b mice as in the others. Experiments with T cell deprived mice show that the adjuvant action of B 19S is thymus-dependent. As the antiSRBC reaction without adjuvant is also thymusdependent, it is difficult to distinguish anti-SRBC and anti-B 19S reactions from the adjuvant action of B 19S on the anti-SRBC reaction. Several explanations are possible, all involving H-2 (Ir?) controlled thymus dependent mechanisms.
It was found that the A/Sn and A.CA strains displayed a strong sensitivity to the well characterized adjuvant action of Brucella (Toujas, 1975), while the A.BY did not. This strain was also found to be very different from the others with regards to the kinetics of splenomegaly following an injection of this adjuvant (Oth, Sabolovic & Le Garrec, 1974). The action of many adjuvants has been shown to be thymus dependent (Allison & Davies, 1971; Del Guercio, 1972). As the genetic difference existing between the presently studied strains lies at the level of the H-2 complex, which contains thymus controlled immune response (Ir) genes (Benacerraf & Katz, 1975), both confirmation of H-2 influence and thymus dependence of the observed adjuvant action were the aims of these studies.
MATERIALS AND METHODS Animals The four congenic resistant strains A/Sn, A.CA, A.SW, A.BY were donated by Dr Eva Klein, Karolinska Institutet, Stockholm. Another congenic resistant pair was used, B 10 and B 10.A. This was a gift of Drs M. and J. Colombani, Hopital St Louis, Paris. The animals were maintained by strict brother x sister mating in our colony, under conventional conditions, in temperature controlled facilities. They were used when 4-8 months old. For a given experiment, they were age, weight and sex matched.
INTRODUCTION We have previously shown a differential adjuvant action of Brucella abortus, as measured by the increase of the number of splenic cells able to form haemolysis 'plaques' on sheep red blood cell layers, in three congenic strains which differ by the H-2 genetic complex (Sabolovic, 0th & Burg, 1971).
Correspondence: Dr Daniel 0th, INSERM U.95 Research Unit, Plateau de Brabois, 54500 Vandoeuvre, France. 0019-2805/78/0500-0807 $02.00 (D) 1978 Blackwell Scientific Publications B
807
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808
Antigens Sheep red blood cells (SRBC) were purchased from the Institut Pasteur Production, Paris. They were washed three times and used as a 10% dilution in saline. Brucella abortus, strain 19 in smooth colonies (B 19S), was heat inactivated (1 h, 650). This was kindly offered by Dr Y. Le Garrec, Service du Professeur Pilet, Ecole Nationale Vt6rinaire, MaisonsAlfort. It was injected as a suspension in sterile saline.
Typing of H-2 haplotype Some (A.CA x A.BY)F2 hybrids were typed for their H-2 haplotype with antisera prepared by multiple intraperitoneal injections of A.CA splenic cells to A.BY and vice versa. The test employed was the haemagglutination technique as described by Kaliss (1973). Plaque forming cells The number of direct plaque forming cells (PFC) was determined exactly as previously (Oth et al., 1974; Sabolovic et al., 1971). Usually, five mice were used for each determination.
Activation of thymus cells Thymus cells were injected intravenously into ten recipients freshly. irradiated with 900 rad X-rays (180 kV, 1 mm Cu filtration) which had been injected 30 min intraperitoneally with 100 i.u. heparin before. Shortly after these treatments, some of the mice received B 19S intraperitoneally, while others didnot. Among B 19S injectedmice, some diedduring the following hours. After 3-5 or 4 days, the mice received intraperitoneally 25 pCi oftritiated thymidine (3H-TdR), purchased from Commissariat a l'Energie Atomique, Gif-sur-Yvette, and with a specific activity of 26-27 Ci/mM. Half an hour after this injection, they were killed, their spleens were removed and dessicated. Radioactivity determinations of the spleens
performed by burning the spleens in a TriCarb 305 sample oxidizer (Packard, Downers Grove, Illinois) and counted in a liquid scintillation counter (type SL30, Intertechnique, Plaisir). The ratio: mean splenic radioactivity of mice injected with B 19S mean splenic radioactivity of non-injected mice was determined for each group.
were
Electrophoretic mobility (E.M.) determination This is determined in the conditions already described (Wioland, Sabolovic & Burg, 1972), using a cylindrical microelectrophoresis apparatus (Mark I, Rank Bros, Bottisham, England). The values are expressed as jpm s-I V-1 cm. Preparation of T lymphocyte 'deprived' mice The procedure described by Davies, Leuchars, Wallis & Koller (1966) was used. 2-month-old mice were thymectomized. 2 months later, they were heavily irradiated with X-rays (850rad, with 1 mm Cu added filtration) and intravenously injected with 5x 106 marrow cells. After a further 3-4 months they were ready to use. Titration of anti-Brucella agglutinins The sera of three to six mice per group were pooled and two-fold diluted in saline, starting from 1/10. Several batches of Brucella test antigens were purchased from Institut Pourquier, Montpellier. Complete agglutination, according to Wright's method, was read after 24 h at 370, in a 1 ml total volume.
RESULTS Confirmation of the dependence on H-2 linked genes of the adjuvant action of B 19S on plaque forming cells It was previously shown (Oth et al., 1974; Sabolovic
Table 1. F2 analysis of the action of the H-2 complex on the adjuvant effect of Brucella the number of SRBC-PFC. (A.CA x A.BY)F2 hybrids are H-2 typed according to haemagglutination test on
H-2 type*
Brucella treatment
H-2t/H-2'
No Yes No Yes
H-2rIH-2r H-2b/H-2b
H-2b/H-2b
PFC per spleen + SE PFC per 106 cells + SE
26,250± 5625t 104,000 ± 20,954 25,468 ±9952 27,812 ± 5
38 0 ±12-5 73-3 ± 11-9 40-1 ±14-6 22-8 ± 5-3
H-2(/H-2b hybrids were used for another purpose. t Mean of two to five mice. *
H-2 linked dependence of adjuvant action of Brucella abortus
809
Table 2. Test of adjuvant effect of Brucella on PFC in BlO.A and BlO mouse strains
Strain
H-2 type
BlO.A
H-2a
B1O
Brucella treatment PFC per spleen ± SE PFC per 106 cells ± SE No Yes No Yes
H-2b
*
64,874 ±28,026*
448,249 + 118,978 54,749 +24,486 103,437 +25,110
252 6 ± 101-5 1220-8 ±676-1 198-4 ± 100-6 184-4 ±45-3
Five mice per group.
al., 1971) that an intravenous injection of 500600 ug of killed B 19S, given 30-32 days before the PFC assay, results in a large increase of PFC numbers in the A/Sn (H-2a) and the A.CA (H-2t) strains, while this is either modest or non-existent in the A.BY (H-2b) strain. To check further the role of the H-2 complex in this difference, we used (A.CA x A.BY)F2 hybrids which were typed by haemagglutination for their H-2 haplotype. They received intravenously 600 pg of B 19S, 27 days before the PFC assay. As seen from the results in Table 1, the A.CA-like H-2f/H-2f mice developed a -4 fold increase in the PFC number, while the A.BY-like H-2 /bH-2b did not demonstrate any increase. The H-2fIH-2b heterozygotes have been used for another experiment and have not been tested here. Previous studies showed that they behave like the A.CA parental strain (Oth et al., 1974). That the H-2a haplotype gives a better adjuvant action of B 19S than the H-2b haplotype was also verified in another genetic background. Mice of the B 10 (H-2b) and B 10.A (H-2a) strains were injected et
with 500 pg B 19S, 34 days before PFC assay. As seen from Table 2, the increase of PFC per spleen was much more pronounced in the H-2' than in the H-26 mice, where no increase of PFC per 106 nucleated cells (spleen cells) was (again) observed. So both these results strongly argue in favour of the importance of the H-2 haplotypefor the presently studied adjuvant action.
Comparison of in vivo thymus cell activation by B 19S, in the A/Sn A.CA, A.SW and A.BY strains Although the Brucella antigens have been considered as thymus-independent antigens (Takahashi, Mond, Carswell & Thorbecke, 1971) certain results suggest some thymus dependence of the maturation of cells sensitive to high levels of these antigens (Gras, Morros, Guix & Tuset, 1975) and a strong activation of thymus cells has been demonstrated (Krueger & Gershon, 1972). We therefore injected intravenously 3-5 x 107 syngeneic thymus cells into lethally irradiated recipients. Within 1 h after irradiation,
Table 3. Activation by B 19S of thymus cells injected in lethally irradiated mice
Strain (H-2 haplotype)
No. of injected thymocytes
Amount of B 19S injected after irradiation (mg)
A/Sn (a)
5 x 107 3 x107 5 x 107
2-5t
A.CA (f)
3x107 A.SW (s)
5x 107
3x107 A.BY (b)
5 x 107
3xl10 * Separating
1-5 2-5 15 2-5 15 25 1-5
Time interval* (days)
3.5 4-0
3-5 40 3-5 4-0 3-5 4-0
irradiation and killing.
t Spleen radioactivity of a 19S injected mice Spleen radioactivity of non-injected mice
t Mortality before 3.5 days was frequently observed with this dose.
Ratiot
4.3, 4-4 2-3 4-1, 6-9 3-1 5-2 6-2 0-8, 2-9,
1-3
1*0
810
D. 0th & D. Sabolovic
some of the treated mice received intraperitoneally 1 5-2 5 mg of the B 19S preparation, while some other remained uninjected. After 3 -5-4 0 days, all the mice received an injection of 3H-TdR, and the ratio: spleen radioactivity of Brucella injected mice spleen radioactivity of non-injected mice was determined. Several experiments were performed, using three to five mice per group each time. The results in Table 3 show that this ratio is always lower in the A.BY mice than in the three other strains, in different experimental conditions. This would suggest that the thymus cell activation by Brucella, as defined by Krueger et al. (1972) is lower in theformer strain than in the three others.
moving B lymphocytes and fast moving T lymphocytes (Wioland et al., 1972). In the case of nontreated mice belonging to the strains A/Sn, A.CA and A.BY, the values of E.M. of both B and T lymphocytes are very similar, varying from 0-68+ 0 01 to 0-69±0-01 for the B cells, and from 1-14+ 0 01 to 1-16±0-01 for the T cells (Donner & Wioland, 1976). If the B 19S is injected a few days before, one observes a dramatic change in the E.M. of A/Sn or A.CA spleen cells, characterized by a diminution of the E.M. of the T cells and an aug-
Differential effects of B 19S on the electrophoretic mobility of spleen cells of A.BY and the other strains Electrophoretic mobility determination of lymphocytes permits a very accurate distinction of slow T
B
A.BY
151
1-
0
z
U
0
z
T
B
A/Sn 15_ I
4
I
10 5
nl
1.5 i-0 0-5 E.M. (Am s"' VW'cm) Figure 1. Histogram of E.M. (number of cells as a function of electrophoretic mobility value of each individual cell) of the lymphoid spleen cells of A.BY (upper) and A/Sn (lower) mice, 8 days after intravenous injection of 200 pg of B 19S. The arrows indicate the mean values for B and T cells of
non-treated mice.
E.M. (pLm s-1 V'1 cm) Figure 2. Histogram of E.M. of spleen cells of A.BY (dotted line) compared with splenic cells of A/Sn (upper), A.CA (middle) and A.SW (lower) (continuous lines) 10 days after i.v. injection of 600,ug of B 19S. The arrows indicate the mean values for B and T cells of non-treated mice.
H-2 linked dependence of adjuvant action of Brucella abortus mentation of that of B cells. Several experiments of that sort have been performed, also using the Peyer patches lymphoid cells as a test for the sensitivity to B 19S injection (to be published). In this regard, the A.BY strain is much less sensitive than the other tested strains. A typical experiment is shown in Fig. 1, where the E.M. pattern of spleen cells of A/Sn and A.BY have been determined 8 days after an intravenous injection of 200 ug of B 19S. As the T cells E.M. of the A/Sn is reduced to 1 -05 ± 0 06, that of the A.BY remains almost unchanged, with a value of 1-13±0-07. Although the E.M. of the A/Sn B cells is augmented to the value of 0-86+0-079 that of the A.BY is less so, with a value of 0-75 ±0 1. Similar results have been obtained comparing A/Sn and A.BY mice, 10 days after 200 ug of B 19S (not shown). Using a larger amount of B 19S, e.g. 600 pg and measuring the E.M. after 10 days, the diminution of the E.M. value of the T cells is more dramatic, except for the strain A.BY. Fig. 2 shows such an experiment, comparing A/Sn, A.CA, A.SW and A.BY strains. However, with such an amount of
811
B 19S, the reaction seems to involve both B and T cell populations with more intensity than with 200 pg. One observes fluctuations in the cell E.M. distribution also in the A.BY strain, but the slowing of the T cells (right on the histogram) is always more pronounced in A/Sn, A.CA or A.SW (continuous line) than in the A.BY (dotted line). One observes a relative quantitative diminution of the slow B cell population after B 19S injection. This is in agreement with the observation that the proportion of immunoglobulin positive lymphocytes (mostly B cells) diminishes after such injections (Dumont, personal communication). Thus, using this criterion, the A.BY seems also to react less in vivo to Brucella antigens than to the A/Sn or A.CA and A.SW, particularly at the level of the T cells. This difference seems rather qualitative than quantitative, because the number of T cells in the A.BY is of the same order of magnitude as in the other strains: data in Tables 1 and 4 permit one to calculate that the number of nucleated spleen cells is the same in the four strains, after an injection of B 19S, and the A.BY has the same B/T
Table 4. Test of adjuvant effect of Brucella on PFC in T cell deprived and control mice of the four congenic resistant strains
Strain A/Sn
Status of mice
Brucella treatment*
Sham
No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes
Deprived A.CA
Sham
Deprived A.SW
Not treated:
Sham
Deprived A.BY
Sham
Deprived
PFC per spleent ± SE
61,249 + 37,920 212,812 ± 16,875 833 ±551 312±312 12,604 + 6988 11,718 +2656 1354 ± 375 1875 4218 ±3906 43,906 ±4844 8734 ±4377 17,812 1406 ±469 2187 7187 ±5013 3624 ±794 1562 ± 1097 937±127
PFC per 106 cells ± SE 222 ± 160 330 ±43 1-7 ±0-8 0-2±0-2 25 ± 13 10 ±0 4 6 ± 1-8 2-8 14-8 ± 14-1 50 3 ± 10-5 30 ± 19 47 4-6 ±2 7-9 12 5 4 1 39±I
2-7±09
500 pg of B 19S injected intravenously 26 days before the PFC determination. to five mice, except where individual value given (one mouse). $ This group was added to confirm that Brucella has an adjuvant effect on PFC in normal mice, as previously found with A/Sn and A.CA. Their age is 6 months.
t Two
D. 0th & D. Sabolovic
812
cell distribution as the A/Sn and A.CA (Donner & Wioland, 1975, 1976). Thymus dependence of the adjuvant effect of B 19S on PFC The previous results suggest that the A.BY strain does not respond to the B 19S adjuvant action because of an H-2 linked defect of thymus sensitivity to these antigens. This was checked by comparing the adjuvant action in 8-month-old male mice belonging to the four congenic strains, in the presence and absence of T lymphocytes. T cell 'deprived' mice were obtained according to Davies et al. (1966) (see Material and Methods) and compared with sham-operated, T cell containing partners. Normal, non-operated A.SW male mice were also responsive to the adjuvant effect of B 19S, as this was previously shown to be the case with A/Sn and A.CA mice (Sabolovic et al., 1971). The results in Table 4 show that the T cell deprived mice of the four strains behave approximately in the same way: not only is the number of PFC dramatically diminished in the deprived mice, but the adjuvant effect of B 19S is completely abolished in all cases. Among the sham-operated (control) mice, the absence of adjuvant effect found in the case of A.BY
is expressed by not only an absence of augmentation of PFC, but by a decrease of this number. In the case of the A.CA, there is no change of the PFC number after B 19S, contrary to what happened previously with younger, non-treated mice (Oth et al., 1974; Sabolovic et al., 1971). On the other hand, sham-operated A/Sn and A.SW strains displayed sensitivity to the adjuvant effect of B 19S. This effect seems nevertheless much more expressed in the non-treated than in the sham-operated mice, as seen from the data obtained with A.SW. Table 4.
Comparison of anti Brucella agglutinins in the four congenic strains Production of anti Brucella agglutinins is a thymus independent event (Takahashi et al., 1971). We therefore wished to measure the total anti-B 19S agglutinins in the four congenic resistant strains. This was performed in age and sex matched mice, using different batches of Brucella test antigens (Table 5), as a function of the time after a single injection, or as a function of the amount of B 19S of one injection, after 32 days (Table 6). The results show that, in this case, the A.BY strain is certainly not a weaker reacter than the three others. On the contrary, it is often the most, or among the most, reactive strains,
Table 5. Reciprocal of the anti Brucella agglutinin titre as a function of the time following an intravenous injection of 500 pg of B 19S
Strain
Day 6
Day 12
Day 23
Day 40
Day 51
Day 75
Day 100
A/Sn A.CA A.SW A.BY
40 40 40 80*
160 160 160 320*
320 320 160 640*
320* 320* 80 320*
320 320 80 640*
40 20 80* 80*
0 20 0 40*
*
Highest value, or values, of the group.
Table 6. Reciprocal of the anti Brucella agglutinin titre 32 days after tion of B 19S, as a function of the amount of B 19S injected
Strain
A/Sn A.CA A.SW A.BY
an
intravenous injec-
250 pg
500 pg
850 pg
1000 pg
1250 pg
1500 pg
1750 pg
80 80 40* 160
160 160 80* 160
640 640 320* 640
640 640 640 640
1280 1280 320* 1280
320 160 80* 320
1280 1280 640* 1280
*
Lowest value of the group.
H-2 linked dependence of adjuvant action of Brucella abortus giving the earliest production of agglutinins and persisting for the longest time. Curiously, the A.SW strain, which has quite normal thymus dependent reactions, often displays a weaker production of agglutinins. This could be correlated with their relative poverty in B lymphocytes, as compared with the other strains (Donner & Wioland, 1975, 1976). The results suggest that the A.BY strain's defective reaction to B 19S is only restricted at the level of the thymus dependent events, but not at the level ofthe thymus independent reactions.
DISCUSSION The marked genetic influence of the genome, on the sensitivity of the adjuvant effect of B 19S on anti SRBC reaction, seems to be closely associated with the H-2 haplotype of the tested mice. This is confirmed in the present work with congenic mouse strains having two different genetic backgrounds, and also with H-2 typed F2 hybrids. Thus, the action of a non H-2 part of the genome on this phenomenon seems improbable. Genetic considerations suggest that the I region of the H-2 complex would be implicated, with a possible complementation effect (Melchers & Rajewsky, 1975) between the I-B and I-C subregion associated genes (unpublished observation). It is therefore tempting to attribute the H-2 influenced adjuvant effect of B 19S on anti SRBC reaction to Ir genes, which have been shown to control anti SRBC reactivity also in the absence of B 19S (Sabolovic et al., 1971). The question is now: wha tare the basic mechanisms of such a control? It must be considered that B 19S, as an adjuvant, is also strongly antigenic by itself. Also, both B 19S, as well as SRBC, are not simple antigens (Le Garrec, 1973). The Ir control of these several antigens could be exerted by several Ir genes, and the overall reaction observed (i.e. adjuvanticity, or absence of adjuvanticity) could result from interactions between these antigens and different Ir controlled reactions. Absence of an adjuvant effect observed with the H-2b haplotype could result from several mechanisms, including non-reactivity, antigenic competition, development of suppressor cells. The H-2b haplotype bearing mouse could be a 'poor responder' to those antigens present in B 19S, which are thymus dependent and able to stimulate an anti SRBC reaction non-
813
specifically, for instance by eliciting the synthesis of PFC stimulating factors (Mond, Takahashi & Thorbecke, 1972). On the other hand, as the H-2b mice are also poor responders to SRBC (Sabolovic et al., 1971) the observed results could be the consequence of a lower thymus-dependent reactivity of H-2b towards SRBC, and not necessarily of their observed poor thymus-dependent reaction against B 19S. With the present results, it is not possible to distinguish clearly if the observed facts result from the reactivity towards the tested antigen SRBC alone, or towards the adjuvant B 19S. On the other hand, the H-2b bearing animals were found to be quite reactive against the B 19S antigens when a thymus-independent reaction was studied. They also might be reactive to thymus-dependent antigens of B 19S, but in such a way that this reaction would result in either suppressive cells (which would block both anti B 19S and SRBC reactions), or in antigenic competition between different antigens contained in B 19S, or in both B 19S and SRBC (Toujas, Dazord & Guelfi, 1974). So, the balance between competition of potentiation of immune reactions against several antigens could be H-2 dependent, explaining the present results. Analysis of these individual reactions should be made, testing as 'adjuvant' different purified antigens of the B 19S preparation. A better characterization of the genetic dependence of the adjuvanticity of a B 19S preparation could be of practical use, because this adjuvant may exert some therapeutic or prophylactic action against some tumours, in some (but not all) strains of mice (Le Garrec, Sabolovic, Toujas, Dazord, Guelfi & Pilet, 1974).
ACKNOWLEDGMENTS We wish to thank Dr Yvonne Le Garrec and Dr Louis Toujas for continued interest, suggestions and discussion of the paper, and Mrs Marie Chantal Beugnot, Mr Pascal Mouchette and Mr Andre Liegey for technical assistance. The work was supported by the INSERM Paris, contract ATP 11-7432. REFEREN CES ALLISON A.C. & DAVIES A.J.S. (1971) Requirements of
thymus dependent lymphocytes for potentiation by adjuvants of antibody formation. Nature (Lond.), 233, 330.
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BENACERRAF B. & KATZ D.H. (1975) The histocompatibilitylinked immune response genes. Advanc. Cancer Res. 21, 21. DAVIEs A.J.S., LEUCHARS E., WALLIS V. & KOLLER P.C. (1966) The mitotic response of thymus-derived cells to antigenic stimulus. Transplantation, 4, 438. DEL GUERCIO P. (1972) Effect of adjuvants on the antibody response to a hapten on a thymus independent carrier. Nature (New Biol.), 238, 213. DONNER M. & WIOLAND M. (1975) Relationship between H-2 region and surface characteristics of the mouse spleen cells. Folia biol. (Prague), 21, 146. DONNER M. & WIOLAND M. (1976) Possible involvement of H-2 region in distribution control of T and B lymphocytes in spleen and in surface characteristics of T lymphocytes. Folia biol. (Prague), 22, 51. GRAS J., MORROS J.M., Guix J. & TUSET N. (1975) Stimulation of IgG antibody formation by sublethal irradiation during persistently repeated immunization with Brucella abortus. Immunology, 28, 629. KALISS M. (1973) An improved polyvinylpyrrolidone (PVP) method for titering mouse allohemagglutinins. Transplantation, 15, 251. KRUEGER J. & GERSHON R.K. (1972) DNA synthetic response of thymocytes to a variety of antigens. J. Immunol. 108, 581. LE GARREC Y. (1973) Variation lisse-rugueuse chez Brucella abortus: quelques aspects immunologiques et immunochimiques. These d'Etat Pharmacie, Universit6 de ParisSud. LE GARREC Y., SABOLOVIc D., ToUJAs L., DAZORD L., GUELFI J. & PILET C. (1974) Activity of inactivated
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