CELLULAR
IMMUNOLOGY
H-2-Linked
48,
249-257
(1979)
Genetic Control of the Plaque-Forming Response to Sheep Red Blood Cells1 DONALD
Transplantation
Cell
M. SILVER
Unit, General Surgical Services, and the Department of Surgery, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114 Received November 22, 1978
The role of genes linked to the H-2 locus in effecting an immune response to SRBC was examined using strains of mice which diier in the classes of antibodies produced following multiple injections with SRBC. While H-2-linked gene action appeared to be at the level of regulating the number of plaque-forming cells (PFC) present in the spleens of different strains following two injections with SRBC, non-H-Zlinked immune response genes seemed to determine whether an IgM-IgG switch occurred as well as how much of each antibody class was produced by the number of PFC available as a result of H-tlinked gene intervention. Mapping studies showed that the H-2-linked genetic effects were due to either the requirement for two genes or the presence of genes located between I-B and H-2D.
INTRODUCTION Regulation of the immune response in mice to sheep erythrocytes (SRBC) is under polygenic control. A study of selectively bred high and low responder lines has shown that the quantitative anti-SRBC agglutinin synthesis is regulated by 7- 13 independently segregating loci (1). Genetically controlled variations in the classes of antibodies produced in response to SRBC have also been described (2-5). The serologic activity of C57BL mice that received multiple injections of SRBC is attributable almost exclusively to IgM antibodies; in hyperimmunized A/J mice the activity of sera is associated largely with IgG molecules. These differences in responsiveness are under the control of three to six independently segregating loci. Thus quantitative and qualitative differences in the immune response to SRBC appear to be under multigenic control. Over the past few years the role of IR genes linked to the H-2 locus in mice in the immune response to a variety of antigens has been studied (6). Because SRBC may contain a large number of antigenic specificities, it has been difficult to evaluate the role of these H-Zlinked genes in the responsiveness to SRBC. Although indications were that 20% of the quantitative differences between high and low responder lines correlated with the H-2 locus, there was no clear role for these genes in effecting the IgM-IgG differences found between C57BL and A/J mice. In this study data will be presented to show that the genes linked to the H-2 locus can affect the plaque-forming celI (PFC) memory response to SRBC in C57BL and 1 This work was supported by USPHS Grant AI-13984. 249
000&8749/791140249-09$02.00/O Copyright AU rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
250
DONALD
M.
SILVER
A/J mice but that control of the proportions of IgM and IgG produced in response to SRBC are attributable to non-H-2-linked genes. MATERIALS
AND METHODS
Animals
Female mice of the inbred strains C57BW6, A/J, BlO.A, BlO.D2(old), BlO.A(2R), BlO.A(5R), and A.BY and female B6AFl hybrids were purchased from Jackson Laboratories, Bar Harbor, Maine. Antisera
Mice were injected intraperitoneally (ip) with 0.1 ml of a 2% suspension of SRBC in 0.9% saline. The SRBC were obtained from the same animal for all experiments, collected in sterile Alsever’s solution, and stored at 4°C for 1 week before use. For injection, the cells were washed three times with saline and resuspended at a concentration of 4 x IO* cells/ml (2%). The mice were injected at weekly intervals, and bleedings made 6 days after a particular injection. Serology
The assay for hemolysins and hemagglutinins was carried out with microtiter plates (Cooke Engineering Co., Alexandria, Va.). For hemagglutination immune plasma which had been diluted 1:5 in saline was added in volumes of 0.025 ml to an equal volume of 0.9% saline or 0.1 M 2-mercaptoethanol(2-ME) in 0.9% saline. The mixtures were incubated at 37°C for 30 min after which time twofold serial dilutions were made in saline using the microtiter apparatus, and 0.025 ml of a 2% suspension of SRBC was added to each well. The plates were then allowed to stand at room temperature for 2 hr after which they were examined for evidence of agglutination. The titer was taken as the reciprocal of the highest dilution of antiserum in which the SRBC showed a settling pattern distinctive from that observed in wells that did not contain antiserum. For determination of hemolysins all reagents were prepared in Veronal-buffered saline (VBS), pH 7.4. Immune plasma was mixed with VBS or 0.1 M 2-ME as for hemagglutination and serial twofold dilutions were made in volumes of 0.025 ml. To each dilution was added 0.025 ml of guinea pig complement which had been diluted 1: 10 and 0.025 ml of a 2% suspension of SRBC. The plates were incubated at 37°C for 30 min and centrifuged at 1000 t-pm for 1 min after which time the mixtures were read for hemolysis. The titer was taken as the highest dilution of serum in which there was clear evidence of hemolysis. The reproducibility of the tests was measured by repeating assays for a single antiserum in the presence or absence of 2-ME. Of nine assays, six gave identical titers both before and after treatment with 2-ME. In three cases a difference of one dilution was recorded in the endpoints. It has been shown (2) that in this system the activity of IgM is sensitive to the action of 2-ME, while the activity of IgG is resistant to 2-ME. Spleen Cells
Mice were sacrificed by cervical dislocation. prepared by perfusing the excised spleens
Spleen cell suspensions with 2 ml of Medium
were L-15
GENETIC
CONTROL
OF THE PFC RESPONSE TO SRBC
251
(Microbiological Associates, Inc., Bethesda, Md.) and then gently teasing them apart with 22-gauge needles. The 2-ml suspension was transferred to a test tube and incubated at 37°C for 1 min, after which time the supematant was removed, transferred to a second tube, and placed at 4°C. Nucleated cells were counted. Plaque Assay
The number of PFC was measured according to a modification of the method of Cunningham and Szenberg (7). Two pieces of “double-sided” tape each 0.25 in. wide were laid along the l-in. sides of clean 3 x l-in. microscope slides, leaving 2.5 in. remaining to be divided up into three areas by two more pieces of 0.25-in. tape. Another slide of the same size (without tape) was pressed firmly onto the tape, forming three chambers which could be filled with a monolayer cell suspension. The number of plaques was recorded as the total per slide. All slides were prepared in duplicate and any slide accepting more than a monolayer cell suspension was discarded. Spleen cells were diluted in Medium L-15 to a concentration of 4 X lo6 cells/ml. To 0.5 ml of this suspension was added 0.05 ml SRBC (final concentration 4 x 108 cells/ml) followed by 0.05 ml complement (1: 13 final concentration of pooled guinea pig serum). For direct PFC measurements, 0.05 ml of medium was added to bring the final volume to 0.65. This mixture was applied with a Pasteur pipet to duplicate slides each receiving a volume of 0.150 ml. The chambers were sealed with heated paraffin-Vaseline and the slides were incubated for 45 min at 37°C. The same procedure was carried out for each spleen to detect indirect PFC, but in place of 0.05 ml of medium 0.05 ml of a goat anti-mouse IgG heavy-chain antiserum diluted 1:8 in L-15 (final concentration 1: 101) was added to the mixture. Immediately after incubation all slides were read microscopically under four-power magnification. In this report direct PFC are referred to as IgM PFC and indirect PFC as IgG PFC. For each spleen the number of direct PFC was subtracted from the total number of plaques developed with the goat antiserum and the result designated as indirect PFC. RESULTS Serological Analysis of the Response to SRBC
Mice of strains C57BW6, A/J, B6AFl hybrids, BlO.A, BlO.A(2R), BlO.A(SR), A.BY, and BlO.D2(old) were injected ip at weekly intervals with 0.1 ml of a 2% suspension of SRBC, and serum was prepared from individual bleedings made 6 days after the fifth injection. These sera were tested for hemagglutinins and hemolysins before and after treatment with 2-ME. Representative results of these tests are shown in Table 1. As reported previously (2), following multiple injections with SRBC, C57BL/6 mice produced antibodies which are highly sensitive to treatment with 2-ME(IgM), whereas the activity of sera obtained from A/J mice that had received the same number of injections showed resistance to 2-ME(IgG). This distinction between the serological responses of C57BL/6 and A/J mice to SRBC are maintained over the dose range 4 x lo6 to 4 x lOa cells (Table 2) and the dose chosen for this study induces the highest responses in both strains. Sera from B6AFl hybrids (Table 1)
252
DONALD
M. SILVER
TABLE
1
Serological Response of Inbred Strains to SRBC” Hemolytic titer Strain
No. of mice
C57BL/6b A/J B6AFl Bl0.A B lO.A(2R) BlO.A(SR) A.BY BlO.D2(old)
25 25 25 25
10 10 15 10
Percentage reduction by 2-ME
Mean
Range
5,218 3,120 5,120
1,280-20,480 640-5,120 1,280- 10,240
2,048 13,120
11,776 4,320 3,093
99 33c 99 93 95 95 48 96
640-5,120
1,280-20,480 2,560-20,480 640-10,240 640-10,240
Agglutinin
972
1,520 836 9,728 173
Percentage reduction by 2-ME
Range
Mean
5,866 1,312 704
titer
95
160-2,560 640-20,480 320-2,560 160-12,830 160-5,120 320-1,280 2,560-20,480 80-320
36 90 35 50 50 37 39
a Mice received five weekly injections of 4 x 10’ SRBC and individual bleedings were prepared 6 days following the fifth injection. b Normal serum controls consisted of prebleeds of 10 C57BL/6 and 10 A/J mice. All sera were negative for hemolysins and hemagglutinins except three sera from C57BL/6 mice which had titers of l/8. These sera lost all activity after treatment with 2-ME. c Since the assay is accurate to within one well, a reduction by 2-ME between 0 and 50% represents the presence of predominantly 2-ME resistant antibodies in the sera.
showed responses indistinguishable from those observed in the C57BW6 parental strain. The immune responses of BlO.A, BlO.A(2R), BlO.A(5R), and BlO.D2 mice were distinctively more sensitive to 2-ME than were sera from A/J mice, although they contained, on the whole, a slightly greater proportion of 2-ME resistant activity than did the sera from C57BL/6 mice. BIO.A is a subline of C57BWlO in which the H-2a complex has been substituted for H-2b, while the BlO.A(2R) and TABLE
2
Serological Responses of C57BL/6 and A/J Mice to Injections of SRBC” Hemolytic titerb Dose (No. of cells)
No. of injections
Mean
Range
Percentage reduction by 2-ME
544 9,728 448 8%
160- 1,280
loo
2,560-20,480 320-640 320-1,280
C57BLl6 4 x 108 4 x 10' 4 x 106 4 x 106
5 5 5 8"
99 99 99
A/J 4 x 108 4 x 10’ 4 x 106 4 x 106
5
5 5 8
400 3,456
80-1,280 640-10,240
120
40-160
176
80-320
50
10 100 50
a Sera were obtained from 10 mice in each group bled 6 days after the last injection. b The titer was taken as the reciprocal of the highest dilution of serum in which there was clear evidence of hemolysis. e C57BW6 mice continue to produce an almost exclusive IgM response even after 15 injections of SRBC at any of the doses specified.
GENETIC
CONTROL
253
OF THE PFC RESPONSE TO SRBC TABLE
3
Alleles of the H-2 Loci under Study Strain
K
I-A
I-B
I-J
I-E
I-C
S
D
C57BLi6 .WJ Bl0.A BlO.A(2R) BlO.A(SR) A.BY B6AFl BlO.D2
b k k k b b b/k d
b k k k b b blk d
b
b k k k k b blk d
b k k k
b d d d d b b/d d
b d d d d b bid d
b d d b d b bid d
k k
k b b b/k d
k
b b/k d
BlO.A(SR) lines show recombination within the H-2 locus (Table 3). When sera from A.BY mice (H-2b is substituted for H-2a, such that these mice are similar, if not identical, to A/J mice except at the H-2 locus) following five injections with SRBC were tested they were found to be resistant to the action of 2-ME, indicating an IgG response to SRBC. Thus the genes within the H-2 complex do not appear to play a decisive role in the response to SRBC, but their participation in some form in the overall responsiveness to that antigen has not been excluded by these data. Role of H-2-Linked Genes in the PFC Response to SRBC
Following multiple injections with SRBC, spleens from C57BL/6 and A/J mice contain IgM and IgG PFC. As shown in Table 4, following the second (and subsequent) injections with SRBC, the average number of IgG PFC (on peak days) in the spleens of A/J mice was many times higher than the number of IgM PFC. In the C57BL/6 mice, however, although the average number of IgM PFC reached a TABLE
4
PFC Response to SRBC: A/J, CS7BLl6, and B6AFl Mice IgM PFC Strain A/J
Day 9
10 11 C57BW6
9
10 11 B6AFl
9
10 11
Secondary 93,000(68-120)” 60,000 25,000 76,000(4346,000 21,000 102,000 105,000(9670,000
IgG PFC Primary 9,000(3-14)
10,000 101)
114)
4,000 22,000(4-27) 8,000 8,000 32,000 28,000(6-35) 14,000
Secondary 434,000 446,000(180-900) 225,000 52,000 108,000(60-140) 62,000 37,000 102,ooo(95-122) 99,000
Primary 60,000 40,000(21-72)
10,000 72,000 100,000(72-130) 59,000 32,000 90,000(7882,000
110)
a Each experiment was performed on three separate occasions and the data were pooled. For each experiment six mice of each strain were immunized on Day 0 with 4 x 10’ SRBC, then on Day 7 three mice were reimmunized and three were not. Days 9, 10, and 11 primary are days 2,3, and 4 secondary. Numbers in parentheses represent the full range of observed value&O3 for the peak day of the secondary response.
254
DONALD
M. SILVER
peak of about 60,000-80,000 PFC per spleen after each injection, it has been shown (3) that the average number of IgG PFC falls from the peak detected on Day 10 following the primary injection (Day 3 following the secondary injection) to 20,000 by the fourth injection with SRBC. Indeed, it is questionable whether there is any IgG PFC response upon secondary stimulation in these mice for when the numbers of IgG PFC detected after one injection with SRBC (days 9, 10, and 11) are subtracted from those detected after a secondary injection on Day 7, few, if any, new IgG PFC remain. All of the IgG PFC detected in the C57BL/6 mice following multiple injections with SRBC can be accounted for by those cells formed exclusively in response to the primary stimulation. Similarly there is no evidence for a secondary IgG PFC response in B6AFl hybrids (Table 4). Data presented in Table 5, however, show an apparent IgG PFC memory response in the spleens of BlO.A, BlO.A(2R), BlO.A(5R), and BlO.D2 mice following two injections with SRBC, if the memory response is defined by the presence of high numbers of IgG PFC relative to those remaining after a primary injection with SRBC. Thus, at the level of the number of PFC present in the spleen following two injections with SRBC, these lines appear more similar to A/J mice than C57BL/6 mice, a finding in contrast with the serological data presented in Table 1. Furthermore, spleens from A.BY mice show a higher number of IgG PFC following two injections with SRBC than can be totally accounted for by those present following the primary SRBC injection, but it is clear that high numbers of these latter IgG PFC remain in the spleens of these mice. A.BY mice respond TABLE
5
PFC Responses to SRBC: BlO.A, BlO.A(ZR), BlO.A(SR), A.BY, and BlO.D2 Mice IgM PFC Strain Bl0.A B lO.A(2R) BlO.A(SR) A.BY BlO.D2
IgG PFC
Day
Secondary
Primary
Secondary
Primary
9 10 11 9 10 11 9 10 11 9 10 11 9 10 11
57,000(36- 110)a 25,000 16,000 52,000(28-74) 16,000 1,500 80,000(62- 140) 25,000 16,000 44,000(32-49) 38,000 12,000 66,000(57-70) 24,000 9,000
7,000(2- 16) 600 1,200 11,000(8- 19) 600 300 28,000(24-40) 1,500 800 1,600(1.1-2.7) 1,200 400 6,600(3- 11) 900 300
210,000 292,000(200-576) 98,000 140,000 280,000(108-434) 64,000 120,000 210,000(86-320) 80,000 140,000 203,000(144-240) 166,000 106,000 198,000(150-245) 56,000
7,000 3,000(0.4-48) 4,000 1,508 3,000(1.2-9) 5,008 26,000 12,000(8-22) 1,700 96,000 140,000(100- 160) 121,000 8,000 22,000(9-38) 3,000
a Each experiment was performed on two separate occasions and the data were pooled. For each experiment six mice of each strain were immunized on Day 0 with 4 x 10’ SRBC, then on Day 7 three mice were reimmunized and three were not. Days 9, 10, and 11 primary are Days 2, 3, and 4 secondary. Numbers in parentheses represent the full range of observed values/lo3 for the peak day of the secondary response.
GENETIC
CONTROL
OF THE PFC RESPONSE
TO SRBC
255
similarly, although not identically, to the C57BW6 strain at the PFC level while serologically they are producing a predominant IgG response. DISCUSSION The immune response to SRBC in mice is a complex phenomenon effected by the intervention of genes at multiple loci. Studies using the high and low responder lines (1) showed regulation of the quantitative anti-SRBC agglutinin response resulting from the interaction of a group of at least 7- 13 independently segregating loci. A definite correlation between H-2 locus and agglutinin titers in these lines was found and there were indications that 20% of the difference in immune responsiveness to SRBC between the high and low responder lines correlated with the H-2 locus. The role played by the intervention of genes linked to the H-2 locus in regulating the immune response to the complex antigen SRBC remains to be determined. Data presented in this study suggestthat one level at which genes linked to the H-2 locus can affect the response to SRBC is the regulation of the expansion of the PFC population following the secondary immunization with SRBC. It is clear, however, that as in the case of the high and low responder lines, such H-2 linked genetic control contributes only in part to the overall control of the anti-SRBC response, with the interaction of non-H-2-linked immune response loci contributing the rest. In response to multiple injections with SRBC, C57BL/6 mice produce IgM almost exclusively, while A/J mice respond predominantly with the production of IgG. B6AFl hybrids and the C57BW6 parent strain responded almost identically and appropriate backcrossanalysis implied three to six independently segregatingdominant loci controlling these diEerences in responsiveness (2). (These loci may be identical with some of those described in the high and low responder lines or they may represent another system of complex control regulating the switchover from IgM to IgG in response to SRBC.) Serological analysis of serafrom Bl0.A and A.BY mice indicated that genes linked to the H-2 locus appeared to play little role in this IgM-IgG difference between C57BL/6 and A/J mice which is detected following immunizations with SRBC . When the PFC response of C57BW6 and A/J mice was examined (3) it was found that a hallmark of the C57BL/6 response was the appearance of IgG PFC following a primary injection with SRBC, which accounted for the IgG PFC present after multiple injections with SRBC. This apparent lack of a secondary IgG PFC was consistent with the serological data which showed that the memory response of these mice would reside in the IgM PFC. It was surprising, therefore, to find that Bl0.A mice, in response to secondary immunization with SRBC, showed high numbers of IgG PFC in their spleens relative to those remaining as a result of primary immunization, when serological data indicated a prolonged IgM response similar (but not identical) to that of C57BW6 mice. Since these two lines differ at the H-2 locus but are otherwise identical at the non-H-2 loci, it appears that the increased number of IgG PFC detected in BIO.A mice resulted from the presence of the genes linked to the H-2” allele derived from A/J mice. That H-Zlinked gene action affected the expansion of the PFC population following secondary immunization with SRBC was confirmed by the data derived from the study of A.BY mice, for in this case while serological data showed a memory IgG response, PFC analysis revealed only a minimal
256
DONALD
M. SILVER
memory response in A.BY mice compared with that of A/J mice (Tables 4 and 5). This would seem due to the presence of the H-2b locus derived from C57BL/6 mice. Using the BlO.A(2R) and BIO.A(SR) recombinant mice, it was shown that the formation of high numbers of IgG PFC following secondary immunization with SRBC was not affected by the presence of a b-allele at H-2K, I-A, I-B, or H-2D. Thus, the gene(s) affecting the PFC response either maps between I-B and H-2D, or the presence of more than one b-allele at two different loci is necessary to produce the effects under study. These effects may reflect a regulatory control exerted by H-Zlinked genes which allow for or suppress the PFC memory response to SRBC. Suppression of the immune response to SRBC must be considered in the light of the fact that the A.BY mice appear to produce some IgG PFC which cannot be accounted for by those remaining from the primary SRBC injection. Further genetic studies are needed to localize the regions of the H-2 locus which control the PFC response to SRBC. It is important to note that the I-J region (to the right of I-B on Table 2) seems to be involved in effecting suppression in a number of systems (12- 15). A conclusion which can be drawn from these data is that while the H-Zlinked gene action described above may be exerted at the level of regulating the secondary IgG PFC response to SRBC, non-H-2-linked immune response genes seem to determine whether an IgM-IgG switchover will occur and how much of each class of antibody will be produced by the number of class-specific PFC available as a result of the H-2-linked gene intervention. Non-H-2-linked genetic control of the quantity of antibodies produced in response to a variety of antigens has been reported (1, 8- 11) and it is clear that in some systems this control is affected by H-Zlinked gene intervention (10, 11). Recently, as a result of in vitro studies performed on the role of mediators in effecting T-B cell cooperation a number of factors have been described (16- 18). Some appear to be coded for by genes linked to the H-2 locus, while others seem unrelated to the gene products of this region. Both types of factors can affect the immune response to different antigens, but each factor may exert its action at different stages in the development of an immune response. The effects of the H-2and non-H-2-linked genes on the in vivo immune response to SRBC discussed above indicate that in vim, genetic control is exerted at various stages in the immune response to SRBC. The data presented above were derived from a study of two inbred strains which differ in their immune responses to SRBC. Recently, Kipps et al. (19) have reported that genes located within the H-2 locus control the magnitude of the PFC response and the heterogeneity of the antibody response to the random linear terpolymer poly(LGl~~~, LLys 36, LAla’O), abbreviated GLA. Although the action of the H-Zlinked genes described above appears to be exerted on the secondary IgG PFC response to SRBC, there may also be control exerted over the IgM memory PFC response (which has been described previously (3) in these same strains) but C57BL/6 and A/J mice may show no variation at these control genes. ACKNOWLEDGMENT I wish to express my gratitude to Marcela Vera-Garcia for her excellent technical assistance.
GENETIC
CONTROL
OF THE PFC RESPONSE
TO SRBC
257
REFERENCES 1. Stiffel, C., Mouton, D., Bouthillier, Y., Heumann, A. M., Decreusefond, C., Mevel, J. C., and Biozzi, G., Progr. Immunol. 2, 203, 1974. 2. Silver, D. M., McKenzie, I., and Winn, H. J., J. Exp. Med. 136, 1063, 1972. 3. Silver, D. M., and Winn, H. .I., Cell. Immunol. 7, 237, 1973. 4. Silver, D. M., and Chai, C. K., J. Immune/. 115, 462, 1975. 5. Seman, M., Chevalier, F., and Stanislawski, M., Eur. J. Zmmunol. 8, 262, 1978. 6. Benacerraf, B., and Katz, D. H., Advan. Cancer Res. 21, 121, 1975. 7. Cunningham, A. J., and Szenberg, A., Immunology 14, 599, 1%8. 8. Dot-f, M. E., Dunham, E. K., Johnson, J. P., and Benacerraf, B., J. Immunol. 112, 1329, 1974. 9. Jormalainen, S., Mozes, E., and Sela, M., J. Exp. Med. 141, 1057, 1975. 10. Silver, D. M., and Lane, D. P., Zmmunogenet. 8, 65, 1979. 11. Pisetsky, D., Berzofsky, J. A., and Sachs, D. H., J. Exp. Med. 147, 599, 1978. 12. Murphy, D. B., Herzenberg, L. A., Okumura, K., Herzenberg, L. A., and McDevitt, H. O., J. Exp. Med. 144,699, 1975. 13. Tada, T., Taniguichi, M., and Takemori, C. S., J. Exp. Med. 144, 713, 1976. 14. Greene, M. I., Pierres, A., Dot-f, M. E., and Benacerraf, B., J. Exp. Med. 146, 293, 1977. 15. Theze, J., Waltenbaugh, C., Dorf, M. E., and Benacerraf, B., J. Exp. Med. 146,287, 1977. 16. Dutton, R. W., Transplant. Rev. 23, 66, 1975. 17. Schimpl, A., Wecker, E., Hubner, L., Hunig, Th., and Muller, G., Progr. Zmmunol. 3,397, 1978. 18. Amerding, D., and Katz, D. H., J. Exp. Med. 137, 547, 1974. 19. Kipps, T. J., Benacerraf, B., and Dot-f, M. E., Eur. J. Immunol. 8, 415, 1978.
IMMUNOLOGY
H-2-Linked
48,
249-257
(1979)
Genetic Control of the Plaque-Forming Response to Sheep Red Blood Cells1 DONALD
Transplantation
Cell
M. SILVER
Unit, General Surgical Services, and the Department of Surgery, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114 Received November 22, 1978
The role of genes linked to the H-2 locus in effecting an immune response to SRBC was examined using strains of mice which diier in the classes of antibodies produced following multiple injections with SRBC. While H-2-linked gene action appeared to be at the level of regulating the number of plaque-forming cells (PFC) present in the spleens of different strains following two injections with SRBC, non-H-Zlinked immune response genes seemed to determine whether an IgM-IgG switch occurred as well as how much of each antibody class was produced by the number of PFC available as a result of H-tlinked gene intervention. Mapping studies showed that the H-2-linked genetic effects were due to either the requirement for two genes or the presence of genes located between I-B and H-2D.
INTRODUCTION Regulation of the immune response in mice to sheep erythrocytes (SRBC) is under polygenic control. A study of selectively bred high and low responder lines has shown that the quantitative anti-SRBC agglutinin synthesis is regulated by 7- 13 independently segregating loci (1). Genetically controlled variations in the classes of antibodies produced in response to SRBC have also been described (2-5). The serologic activity of C57BL mice that received multiple injections of SRBC is attributable almost exclusively to IgM antibodies; in hyperimmunized A/J mice the activity of sera is associated largely with IgG molecules. These differences in responsiveness are under the control of three to six independently segregating loci. Thus quantitative and qualitative differences in the immune response to SRBC appear to be under multigenic control. Over the past few years the role of IR genes linked to the H-2 locus in mice in the immune response to a variety of antigens has been studied (6). Because SRBC may contain a large number of antigenic specificities, it has been difficult to evaluate the role of these H-Zlinked genes in the responsiveness to SRBC. Although indications were that 20% of the quantitative differences between high and low responder lines correlated with the H-2 locus, there was no clear role for these genes in effecting the IgM-IgG differences found between C57BL and A/J mice. In this study data will be presented to show that the genes linked to the H-2 locus can affect the plaque-forming celI (PFC) memory response to SRBC in C57BL and 1 This work was supported by USPHS Grant AI-13984. 249
000&8749/791140249-09$02.00/O Copyright AU rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
250
DONALD
M.
SILVER
A/J mice but that control of the proportions of IgM and IgG produced in response to SRBC are attributable to non-H-2-linked genes. MATERIALS
AND METHODS
Animals
Female mice of the inbred strains C57BW6, A/J, BlO.A, BlO.D2(old), BlO.A(2R), BlO.A(5R), and A.BY and female B6AFl hybrids were purchased from Jackson Laboratories, Bar Harbor, Maine. Antisera
Mice were injected intraperitoneally (ip) with 0.1 ml of a 2% suspension of SRBC in 0.9% saline. The SRBC were obtained from the same animal for all experiments, collected in sterile Alsever’s solution, and stored at 4°C for 1 week before use. For injection, the cells were washed three times with saline and resuspended at a concentration of 4 x IO* cells/ml (2%). The mice were injected at weekly intervals, and bleedings made 6 days after a particular injection. Serology
The assay for hemolysins and hemagglutinins was carried out with microtiter plates (Cooke Engineering Co., Alexandria, Va.). For hemagglutination immune plasma which had been diluted 1:5 in saline was added in volumes of 0.025 ml to an equal volume of 0.9% saline or 0.1 M 2-mercaptoethanol(2-ME) in 0.9% saline. The mixtures were incubated at 37°C for 30 min after which time twofold serial dilutions were made in saline using the microtiter apparatus, and 0.025 ml of a 2% suspension of SRBC was added to each well. The plates were then allowed to stand at room temperature for 2 hr after which they were examined for evidence of agglutination. The titer was taken as the reciprocal of the highest dilution of antiserum in which the SRBC showed a settling pattern distinctive from that observed in wells that did not contain antiserum. For determination of hemolysins all reagents were prepared in Veronal-buffered saline (VBS), pH 7.4. Immune plasma was mixed with VBS or 0.1 M 2-ME as for hemagglutination and serial twofold dilutions were made in volumes of 0.025 ml. To each dilution was added 0.025 ml of guinea pig complement which had been diluted 1: 10 and 0.025 ml of a 2% suspension of SRBC. The plates were incubated at 37°C for 30 min and centrifuged at 1000 t-pm for 1 min after which time the mixtures were read for hemolysis. The titer was taken as the highest dilution of serum in which there was clear evidence of hemolysis. The reproducibility of the tests was measured by repeating assays for a single antiserum in the presence or absence of 2-ME. Of nine assays, six gave identical titers both before and after treatment with 2-ME. In three cases a difference of one dilution was recorded in the endpoints. It has been shown (2) that in this system the activity of IgM is sensitive to the action of 2-ME, while the activity of IgG is resistant to 2-ME. Spleen Cells
Mice were sacrificed by cervical dislocation. prepared by perfusing the excised spleens
Spleen cell suspensions with 2 ml of Medium
were L-15
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OF THE PFC RESPONSE TO SRBC
251
(Microbiological Associates, Inc., Bethesda, Md.) and then gently teasing them apart with 22-gauge needles. The 2-ml suspension was transferred to a test tube and incubated at 37°C for 1 min, after which time the supematant was removed, transferred to a second tube, and placed at 4°C. Nucleated cells were counted. Plaque Assay
The number of PFC was measured according to a modification of the method of Cunningham and Szenberg (7). Two pieces of “double-sided” tape each 0.25 in. wide were laid along the l-in. sides of clean 3 x l-in. microscope slides, leaving 2.5 in. remaining to be divided up into three areas by two more pieces of 0.25-in. tape. Another slide of the same size (without tape) was pressed firmly onto the tape, forming three chambers which could be filled with a monolayer cell suspension. The number of plaques was recorded as the total per slide. All slides were prepared in duplicate and any slide accepting more than a monolayer cell suspension was discarded. Spleen cells were diluted in Medium L-15 to a concentration of 4 X lo6 cells/ml. To 0.5 ml of this suspension was added 0.05 ml SRBC (final concentration 4 x 108 cells/ml) followed by 0.05 ml complement (1: 13 final concentration of pooled guinea pig serum). For direct PFC measurements, 0.05 ml of medium was added to bring the final volume to 0.65. This mixture was applied with a Pasteur pipet to duplicate slides each receiving a volume of 0.150 ml. The chambers were sealed with heated paraffin-Vaseline and the slides were incubated for 45 min at 37°C. The same procedure was carried out for each spleen to detect indirect PFC, but in place of 0.05 ml of medium 0.05 ml of a goat anti-mouse IgG heavy-chain antiserum diluted 1:8 in L-15 (final concentration 1: 101) was added to the mixture. Immediately after incubation all slides were read microscopically under four-power magnification. In this report direct PFC are referred to as IgM PFC and indirect PFC as IgG PFC. For each spleen the number of direct PFC was subtracted from the total number of plaques developed with the goat antiserum and the result designated as indirect PFC. RESULTS Serological Analysis of the Response to SRBC
Mice of strains C57BW6, A/J, B6AFl hybrids, BlO.A, BlO.A(2R), BlO.A(SR), A.BY, and BlO.D2(old) were injected ip at weekly intervals with 0.1 ml of a 2% suspension of SRBC, and serum was prepared from individual bleedings made 6 days after the fifth injection. These sera were tested for hemagglutinins and hemolysins before and after treatment with 2-ME. Representative results of these tests are shown in Table 1. As reported previously (2), following multiple injections with SRBC, C57BL/6 mice produced antibodies which are highly sensitive to treatment with 2-ME(IgM), whereas the activity of sera obtained from A/J mice that had received the same number of injections showed resistance to 2-ME(IgG). This distinction between the serological responses of C57BL/6 and A/J mice to SRBC are maintained over the dose range 4 x lo6 to 4 x lOa cells (Table 2) and the dose chosen for this study induces the highest responses in both strains. Sera from B6AFl hybrids (Table 1)
252
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TABLE
1
Serological Response of Inbred Strains to SRBC” Hemolytic titer Strain
No. of mice
C57BL/6b A/J B6AFl Bl0.A B lO.A(2R) BlO.A(SR) A.BY BlO.D2(old)
25 25 25 25
10 10 15 10
Percentage reduction by 2-ME
Mean
Range
5,218 3,120 5,120
1,280-20,480 640-5,120 1,280- 10,240
2,048 13,120
11,776 4,320 3,093
99 33c 99 93 95 95 48 96
640-5,120
1,280-20,480 2,560-20,480 640-10,240 640-10,240
Agglutinin
972
1,520 836 9,728 173
Percentage reduction by 2-ME
Range
Mean
5,866 1,312 704
titer
95
160-2,560 640-20,480 320-2,560 160-12,830 160-5,120 320-1,280 2,560-20,480 80-320
36 90 35 50 50 37 39
a Mice received five weekly injections of 4 x 10’ SRBC and individual bleedings were prepared 6 days following the fifth injection. b Normal serum controls consisted of prebleeds of 10 C57BL/6 and 10 A/J mice. All sera were negative for hemolysins and hemagglutinins except three sera from C57BL/6 mice which had titers of l/8. These sera lost all activity after treatment with 2-ME. c Since the assay is accurate to within one well, a reduction by 2-ME between 0 and 50% represents the presence of predominantly 2-ME resistant antibodies in the sera.
showed responses indistinguishable from those observed in the C57BW6 parental strain. The immune responses of BlO.A, BlO.A(2R), BlO.A(5R), and BlO.D2 mice were distinctively more sensitive to 2-ME than were sera from A/J mice, although they contained, on the whole, a slightly greater proportion of 2-ME resistant activity than did the sera from C57BL/6 mice. BIO.A is a subline of C57BWlO in which the H-2a complex has been substituted for H-2b, while the BlO.A(2R) and TABLE
2
Serological Responses of C57BL/6 and A/J Mice to Injections of SRBC” Hemolytic titerb Dose (No. of cells)
No. of injections
Mean
Range
Percentage reduction by 2-ME
544 9,728 448 8%
160- 1,280
loo
2,560-20,480 320-640 320-1,280
C57BLl6 4 x 108 4 x 10' 4 x 106 4 x 106
5 5 5 8"
99 99 99
A/J 4 x 108 4 x 10’ 4 x 106 4 x 106
5
5 5 8
400 3,456
80-1,280 640-10,240
120
40-160
176
80-320
50
10 100 50
a Sera were obtained from 10 mice in each group bled 6 days after the last injection. b The titer was taken as the reciprocal of the highest dilution of serum in which there was clear evidence of hemolysis. e C57BW6 mice continue to produce an almost exclusive IgM response even after 15 injections of SRBC at any of the doses specified.
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253
OF THE PFC RESPONSE TO SRBC TABLE
3
Alleles of the H-2 Loci under Study Strain
K
I-A
I-B
I-J
I-E
I-C
S
D
C57BLi6 .WJ Bl0.A BlO.A(2R) BlO.A(SR) A.BY B6AFl BlO.D2
b k k k b b b/k d
b k k k b b blk d
b
b k k k k b blk d
b k k k
b d d d d b b/d d
b d d d d b bid d
b d d b d b bid d
k k
k b b b/k d
k
b b/k d
BlO.A(SR) lines show recombination within the H-2 locus (Table 3). When sera from A.BY mice (H-2b is substituted for H-2a, such that these mice are similar, if not identical, to A/J mice except at the H-2 locus) following five injections with SRBC were tested they were found to be resistant to the action of 2-ME, indicating an IgG response to SRBC. Thus the genes within the H-2 complex do not appear to play a decisive role in the response to SRBC, but their participation in some form in the overall responsiveness to that antigen has not been excluded by these data. Role of H-2-Linked Genes in the PFC Response to SRBC
Following multiple injections with SRBC, spleens from C57BL/6 and A/J mice contain IgM and IgG PFC. As shown in Table 4, following the second (and subsequent) injections with SRBC, the average number of IgG PFC (on peak days) in the spleens of A/J mice was many times higher than the number of IgM PFC. In the C57BL/6 mice, however, although the average number of IgM PFC reached a TABLE
4
PFC Response to SRBC: A/J, CS7BLl6, and B6AFl Mice IgM PFC Strain A/J
Day 9
10 11 C57BW6
9
10 11 B6AFl
9
10 11
Secondary 93,000(68-120)” 60,000 25,000 76,000(4346,000 21,000 102,000 105,000(9670,000
IgG PFC Primary 9,000(3-14)
10,000 101)
114)
4,000 22,000(4-27) 8,000 8,000 32,000 28,000(6-35) 14,000
Secondary 434,000 446,000(180-900) 225,000 52,000 108,000(60-140) 62,000 37,000 102,ooo(95-122) 99,000
Primary 60,000 40,000(21-72)
10,000 72,000 100,000(72-130) 59,000 32,000 90,000(7882,000
110)
a Each experiment was performed on three separate occasions and the data were pooled. For each experiment six mice of each strain were immunized on Day 0 with 4 x 10’ SRBC, then on Day 7 three mice were reimmunized and three were not. Days 9, 10, and 11 primary are days 2,3, and 4 secondary. Numbers in parentheses represent the full range of observed value&O3 for the peak day of the secondary response.
254
DONALD
M. SILVER
peak of about 60,000-80,000 PFC per spleen after each injection, it has been shown (3) that the average number of IgG PFC falls from the peak detected on Day 10 following the primary injection (Day 3 following the secondary injection) to 20,000 by the fourth injection with SRBC. Indeed, it is questionable whether there is any IgG PFC response upon secondary stimulation in these mice for when the numbers of IgG PFC detected after one injection with SRBC (days 9, 10, and 11) are subtracted from those detected after a secondary injection on Day 7, few, if any, new IgG PFC remain. All of the IgG PFC detected in the C57BL/6 mice following multiple injections with SRBC can be accounted for by those cells formed exclusively in response to the primary stimulation. Similarly there is no evidence for a secondary IgG PFC response in B6AFl hybrids (Table 4). Data presented in Table 5, however, show an apparent IgG PFC memory response in the spleens of BlO.A, BlO.A(2R), BlO.A(5R), and BlO.D2 mice following two injections with SRBC, if the memory response is defined by the presence of high numbers of IgG PFC relative to those remaining after a primary injection with SRBC. Thus, at the level of the number of PFC present in the spleen following two injections with SRBC, these lines appear more similar to A/J mice than C57BL/6 mice, a finding in contrast with the serological data presented in Table 1. Furthermore, spleens from A.BY mice show a higher number of IgG PFC following two injections with SRBC than can be totally accounted for by those present following the primary SRBC injection, but it is clear that high numbers of these latter IgG PFC remain in the spleens of these mice. A.BY mice respond TABLE
5
PFC Responses to SRBC: BlO.A, BlO.A(ZR), BlO.A(SR), A.BY, and BlO.D2 Mice IgM PFC Strain Bl0.A B lO.A(2R) BlO.A(SR) A.BY BlO.D2
IgG PFC
Day
Secondary
Primary
Secondary
Primary
9 10 11 9 10 11 9 10 11 9 10 11 9 10 11
57,000(36- 110)a 25,000 16,000 52,000(28-74) 16,000 1,500 80,000(62- 140) 25,000 16,000 44,000(32-49) 38,000 12,000 66,000(57-70) 24,000 9,000
7,000(2- 16) 600 1,200 11,000(8- 19) 600 300 28,000(24-40) 1,500 800 1,600(1.1-2.7) 1,200 400 6,600(3- 11) 900 300
210,000 292,000(200-576) 98,000 140,000 280,000(108-434) 64,000 120,000 210,000(86-320) 80,000 140,000 203,000(144-240) 166,000 106,000 198,000(150-245) 56,000
7,000 3,000(0.4-48) 4,000 1,508 3,000(1.2-9) 5,008 26,000 12,000(8-22) 1,700 96,000 140,000(100- 160) 121,000 8,000 22,000(9-38) 3,000
a Each experiment was performed on two separate occasions and the data were pooled. For each experiment six mice of each strain were immunized on Day 0 with 4 x 10’ SRBC, then on Day 7 three mice were reimmunized and three were not. Days 9, 10, and 11 primary are Days 2, 3, and 4 secondary. Numbers in parentheses represent the full range of observed values/lo3 for the peak day of the secondary response.
GENETIC
CONTROL
OF THE PFC RESPONSE
TO SRBC
255
similarly, although not identically, to the C57BW6 strain at the PFC level while serologically they are producing a predominant IgG response. DISCUSSION The immune response to SRBC in mice is a complex phenomenon effected by the intervention of genes at multiple loci. Studies using the high and low responder lines (1) showed regulation of the quantitative anti-SRBC agglutinin response resulting from the interaction of a group of at least 7- 13 independently segregating loci. A definite correlation between H-2 locus and agglutinin titers in these lines was found and there were indications that 20% of the difference in immune responsiveness to SRBC between the high and low responder lines correlated with the H-2 locus. The role played by the intervention of genes linked to the H-2 locus in regulating the immune response to the complex antigen SRBC remains to be determined. Data presented in this study suggestthat one level at which genes linked to the H-2 locus can affect the response to SRBC is the regulation of the expansion of the PFC population following the secondary immunization with SRBC. It is clear, however, that as in the case of the high and low responder lines, such H-2 linked genetic control contributes only in part to the overall control of the anti-SRBC response, with the interaction of non-H-2-linked immune response loci contributing the rest. In response to multiple injections with SRBC, C57BL/6 mice produce IgM almost exclusively, while A/J mice respond predominantly with the production of IgG. B6AFl hybrids and the C57BW6 parent strain responded almost identically and appropriate backcrossanalysis implied three to six independently segregatingdominant loci controlling these diEerences in responsiveness (2). (These loci may be identical with some of those described in the high and low responder lines or they may represent another system of complex control regulating the switchover from IgM to IgG in response to SRBC.) Serological analysis of serafrom Bl0.A and A.BY mice indicated that genes linked to the H-2 locus appeared to play little role in this IgM-IgG difference between C57BL/6 and A/J mice which is detected following immunizations with SRBC . When the PFC response of C57BW6 and A/J mice was examined (3) it was found that a hallmark of the C57BL/6 response was the appearance of IgG PFC following a primary injection with SRBC, which accounted for the IgG PFC present after multiple injections with SRBC. This apparent lack of a secondary IgG PFC was consistent with the serological data which showed that the memory response of these mice would reside in the IgM PFC. It was surprising, therefore, to find that Bl0.A mice, in response to secondary immunization with SRBC, showed high numbers of IgG PFC in their spleens relative to those remaining as a result of primary immunization, when serological data indicated a prolonged IgM response similar (but not identical) to that of C57BW6 mice. Since these two lines differ at the H-2 locus but are otherwise identical at the non-H-2 loci, it appears that the increased number of IgG PFC detected in BIO.A mice resulted from the presence of the genes linked to the H-2” allele derived from A/J mice. That H-Zlinked gene action affected the expansion of the PFC population following secondary immunization with SRBC was confirmed by the data derived from the study of A.BY mice, for in this case while serological data showed a memory IgG response, PFC analysis revealed only a minimal
256
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M. SILVER
memory response in A.BY mice compared with that of A/J mice (Tables 4 and 5). This would seem due to the presence of the H-2b locus derived from C57BL/6 mice. Using the BlO.A(2R) and BIO.A(SR) recombinant mice, it was shown that the formation of high numbers of IgG PFC following secondary immunization with SRBC was not affected by the presence of a b-allele at H-2K, I-A, I-B, or H-2D. Thus, the gene(s) affecting the PFC response either maps between I-B and H-2D, or the presence of more than one b-allele at two different loci is necessary to produce the effects under study. These effects may reflect a regulatory control exerted by H-Zlinked genes which allow for or suppress the PFC memory response to SRBC. Suppression of the immune response to SRBC must be considered in the light of the fact that the A.BY mice appear to produce some IgG PFC which cannot be accounted for by those remaining from the primary SRBC injection. Further genetic studies are needed to localize the regions of the H-2 locus which control the PFC response to SRBC. It is important to note that the I-J region (to the right of I-B on Table 2) seems to be involved in effecting suppression in a number of systems (12- 15). A conclusion which can be drawn from these data is that while the H-Zlinked gene action described above may be exerted at the level of regulating the secondary IgG PFC response to SRBC, non-H-2-linked immune response genes seem to determine whether an IgM-IgG switchover will occur and how much of each class of antibody will be produced by the number of class-specific PFC available as a result of the H-2-linked gene intervention. Non-H-2-linked genetic control of the quantity of antibodies produced in response to a variety of antigens has been reported (1, 8- 11) and it is clear that in some systems this control is affected by H-Zlinked gene intervention (10, 11). Recently, as a result of in vitro studies performed on the role of mediators in effecting T-B cell cooperation a number of factors have been described (16- 18). Some appear to be coded for by genes linked to the H-2 locus, while others seem unrelated to the gene products of this region. Both types of factors can affect the immune response to different antigens, but each factor may exert its action at different stages in the development of an immune response. The effects of the H-2and non-H-2-linked genes on the in vivo immune response to SRBC discussed above indicate that in vim, genetic control is exerted at various stages in the immune response to SRBC. The data presented above were derived from a study of two inbred strains which differ in their immune responses to SRBC. Recently, Kipps et al. (19) have reported that genes located within the H-2 locus control the magnitude of the PFC response and the heterogeneity of the antibody response to the random linear terpolymer poly(LGl~~~, LLys 36, LAla’O), abbreviated GLA. Although the action of the H-Zlinked genes described above appears to be exerted on the secondary IgG PFC response to SRBC, there may also be control exerted over the IgM memory PFC response (which has been described previously (3) in these same strains) but C57BL/6 and A/J mice may show no variation at these control genes. ACKNOWLEDGMENT I wish to express my gratitude to Marcela Vera-Garcia for her excellent technical assistance.
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CONTROL
OF THE PFC RESPONSE
TO SRBC
257
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