CELLULAR
IMMUNOLOGY
21,
112-120 (1976)
The Effects of Carrier-Specific Avidity
in the Anti-Hapten
FRED SANFILIPPO Division
of Immunology,
Helper T-Cell
l AND
Duke Medical Received
DAVID
Tolerance
on Antibody
Response W.
Center, Durham,
SCOTT
North
Carolina,
27710
August 11,1975
Rats rendered tolerant to ultracentrifuged sheep -y-globulin (SGG) have been shown to make a poor anti-trinitrophenyl (TNP)-specific antibody response upon challenge with TNP-SGG in complete Freund’s adjuvant (CFA). We have been able to use this carrier-tolerance system in studying specific helper T-cell unresponsiveness to IgG and IgM antibody responses. By using the plaque inhibition technique to measure antibody avidity, we found that there appears to be no difference in the avidity of antibody responses to TNP between the SGG-tolerant and control groups when both are challenged with TNP-SGG in CFA. This was found to be true in both the 19 and 7s antibody responses ia viva as well as in an adoptive transfer model. In addition, studies on the maturation of 19 and 7s antibody responses showed no differences in antibody avidity between carrier-tolerant and control groups. These findings imply that carrierspecific helper T cells do not play a controlling role in determining whether high- or low-avidity hapten-specific B-cell precursors will proliferate in response to challenge with a hapten-carrier conjugate.
INTRODUCTION The process of clonal selection in the antibody response to specific antigens has been extensively examined in recent years, but the exact nature of how high- vs low-avidity clonal precursor cells are stimulated to proliferate remains a subject of current investigation in many laboratories (l-6). Much of this recent work has focused on the plaque inhibition assay of Andersson (7) as a method of studying the selection process of antibody avidity in response to specific antigen (S-10). This work has provided useful information especially regarding the maturation process of the antibody response, although recent controversy has developed around the validity of the plaque inhibition technique itself (11). In this paper we have attempted to examine the role of the carrier-specific helper T cell 2 in the final determination of antibody avidity in response to specific anti1 Predoctoral Fellow in the Medical Scientist Training Program supported by USPHS Grant No. GM 01678. This publication is also supported in part by USPHS Grant No. A110716. a Abbreviations used: SGG, sheep r-globulin;TNP, trinitrophenyl ; CFA, complete Freund’s adjuvant; T cell, thymus cell ; B cell, bone-marrow-derived cell; PFC, plaque-forming cell; SGGSo, soluble sheep y-globulin; KLH, keyhole limpet hemocyanin; RbGG, rabbit y-globulin; PBS, phosphate-buffered saline ; FCS, fetal calf serum; GRBC, goat erythrocytes ; Ig, immunoglobulin ; DNP, dinitrophenyl ; BGG, bovine y-globulin. 112 Copyright All rights
1976 by Academic Press, oP reproduction in any form
Inc. reserved.
HELPER
T
CELLS
AND
ANTIBODY
113
AVIDITY
gens from the standpoint of carrier-specific helper T-cell tolerance. While it has proven difficult to study selective cell populations in Z&JO, we have been able to study helper T cells by using a carrier-specific tolerance model. By inducing tolerance to a soluble protein, it has been shown that a marked reduction is seen in the subsequent antibody response to a hapten conjugated to the original protein (12-15). Using soluble SGG (SGGSo) as carrier tolerogen and TNP-SGG in CFA as the hapten-carrier conjugate, we have studied this phenomenon in detail, and our findings to date strongly suggest that this system provides a mechanism for studying tolerance of specific helper T cells involved in IgG, IgM and IgE responses (16-19). In this report we have utilized this system to examine the effects of carriertolerant vs normal immune helper T cells on the avidity of hapten-specific antibody responses to hapten+arrier conjugates. Our data suggest that helper T cells are not involved in the final determination of antibody avidity.3 Our incidental findings also confirm recent work by others (S-11) revealing certain ambiguities in the plaque inhibition technique as a method for direct determination of antibody affinity.3 MATERIALS
AND METHODS
A&n&. Eight- to ten-week-old inbred male Lewis rats were purchased from Microbiological Associates (Bethesda, Md.). In each experiment only age- and sex-matched rats were used. Tolerogen. Ultracentrifuged SGGSo was prepared as described previously (20). This tolerogen was prepared separately for each experiment from a stock solution, centrifuged at 100,OOOgat 4°C for 60 min without braking. Only the upper onethird of the solution was used, and a dose of 10 mg in a l-ml volume was injected iv for each rat tolerized. Rats were tolerized 1 day before challenge with haptencarrier complexes. Inznzunogen. Trinitrophenylated sheep y-globulin (TNP-SGG) was prepared as described earlier (17) and emulsified with an equal volume of complete Freund’s adjuvant (Difco) to a final concentration of 1 mg of TNPsSGG/ml in CFA. Tolerant and control rats were challenged with O.l-ml amounts of this immunogen intradermally in both hind footpads for a total dose of 200 g/rat, except where otherwise noted. TNP-conjugated proteins. TNPsSGG, TNP&GG, TNP,,KLH and TNPZ2RbIg were prepared by reacting trinitrobenzene sulfonic acid with the appropriate amount of protein in cacodylate buffer as described previously (17, 21). The final TNP to protein ratios were determined by standard techniques (22, 23) and millimolar dilutions on the basis of total TNP were made in PBS. TNP-p-alanine and r-TNP-L-lysine were purchased from Nutritional Biochemicals, Cleveland, Ohio. Preparation of cell suspensions. Popliteal lymph nodes were removed at various times after challenge and cells were collected in Medium 199 with 570 FCS by gentle agitation through a stainless-steel Susi press (Wm. F. Mayer Co., Yonkers, N.Y.). Cells were washed once and resuspended in Medium 199 with 5% FCS and kept at 4°C until use, usually within 30 min. Plaque inhibition assay of PFC. Popliteal lymph node cells were assayed for plaque-forming cells against TNP-goat red blood cells (TNP-GRBC) which were s Avidity
is defined as the tendency
of an antibody
to form
stable complexes
with
molecular antigen. Affinity is the average binding constant of antibody to antigen.
a macro-
114
SANFILIPPO
AND
SCOTT
prepared by the method of Rittenberg and Pratt (21). The plaque assay used was a modified Jerne plate method as earlier described in detail (24). Indirect (IgG) PFC were developed with a 1: 200 dilution of rabbit anti-rat y-globulin. Inhibition with TNP compounds (TNP-Ala, TNPsSGG, TNPs,,RbGG and TNPssSGG) was carried out by adding the appropriate concentration of inhibitor to the agar overlay tubes. Relative avidity was expressed as the percentage of inhibition of total PFC/rat in each group vs the concentration of TNP inhibitor. Control PFC for each group was the total number of PFC/rat without treatment with inhibitor. For tolerant groups the PFC value was always significantly lower (80%) than control immune groups, and plaque inhibition was calculated as the percentage of reduction from the initial PFC/rat. High-avidity antibody is presumably saturated at a lower concentration of TNP inhibitor than low-avidity antibody, so that low concentrations of the TNP inhibitor block high-avidity antibody from lysing TNP-GRBC (7, 9, 2.5). While avidity measurements using this technique are often expressed only in terms of the Iso point of plaque inhibition, i.e., the concentration of hapten which inhibits the number of PFC by 507 O, we have chosen to present the entire inhibition curve as a measure of avidity. TNP plaque inhibition by this technique was shown to be specific since anti-GRBC PFC were not inhibited by any concentration of any TNP compound. Presentation of data. Groups of three rats were used in each experiment and assays were done in duplicate. Values are expressed as the arithmetic mean per group t standard error. RESULTS Avidity
of Anti-TNP
Antibody
in Carrier
(Helper
T-Cell)-Tolerant
vs Control
Groups
Our previous work in this system has already established various parameters of carrier-specific tolerance (16-19). Briefly, rats pretreated iv with 10 mg of SGGSo and challenged 1 day later with TNP-SGG in CFA show a significant reduction in anti-TNP PFC. This carrier tolerance has been shown to reflect specific unresponsiveness in helper T-cell populations involved in IgM and IgG antibody responses and is apparently not a result of suppressor T-cell activity (16). Figures la and b show that tolerance to SGG does not appear to affect either IgG or IgM anti-TNP antibody avidity in response to, challenge with TNP-SGG. In the first experiment, direct (IgM) PFC (Fig. la) were determined 6 days after challenge with 100 pg of TNP-SGG in CFA. At this time and with this challenge dose, the IgM anti-TNP response in the control carrier-tolerant group is 16% of the control nontolerant group, i.e., 84% tolerant. Both groups, however, show an identical percentage of reduction in total PFC/rat upon plaque inhibition with 1O-8-1@* M TNP. Figure lb shows that the avidity of the IgG response at day 20 after challenge with 3 pg of TNP-SGG is also unaffected by carrier tolerance. In this second experiment the control carrier-tolerant group showed a 94% decrease in anti-TNP PFC as compared to the nontolerant control group. In both experiments TNPsSGG was used as PFC inhibitor. To determine whether the avidity of anti-hapten antibody was affected by carrier tolerance as the IgG response matured, these experiments were repeated at days 20 and 29 after challenge. In addition, challenge doses were varied from 3 to 100
HELPER
I
T
CELLS
AND
b-4
ANTIBODY
NORMAL CARRIER
TOLERANT (b)
TNP
11.5
AVIDITY
CONCENTRATION
IyG
PFC AT DAY 20
(M)
FIG. 1. Effect of carrier-specific (helper T-cell) tolerance on anti-hapten antibody avidity. Carrier-tolerant groups were pretreated with 10 mg of SGGSo 1 day prior to challenge with TNP-SGG in CFA. Anti-TNP antibody avidity is expressed as percentage of TNP PFC inhibition vs concentration of TNP as TNPSGG inhibitor, with 1, points indicated by arrowhead. (a), Comparison of IgM anti-TNP plaque inhibition of carrier-tolerant and control groups at day 6 after challenge. (b), Comparison of IgG anti-TNP avidity of carrier-tolerant and control groups at day 20. In both experiments, anti-TNP antibody was significantly reduced in tolerant groups, but, as the graphs show, there were no apparent differences in avidity.
pg of TNP-SGG, and in all cases there were no significant differences in avidity between the carrier-tolerant and nontolerant groups. Preliminary findings also indicate that the avidity of anti-TNP antibody in the secondary response is also unaffected by carrier tolerance but that rats made hapten (B cell) tolerant by pretreatment with TNP-Rat Ig showed a significant reduction in anti-TNP avidity (data not shown). Avidity
of Carrier-Tolerant
vs Control Groups in Adoptive
Transfer
In order to rule out the possibility that carrier-specific tolerance in our in vivo system is merely due to an ongoing competition between tolerogen and immunogen or that the carrier determinants of the hapten-carrier immunogen are blocked by undetectable levels of anti-carrier antibodies responding to the tolerogen, we have carried out several studies using an adoptive transfer model. Briefly, normal donor rats or rats made tolerant to carrier protein 1 day before transfer are sacrificed, their spleen and lymph node cells pooled in separate groups and, after thorough washing, cells are transferred iv into lethally irradiated syngeneic recipients. Using this adoptive transfer model, we have been able to show that such carrier tolerance can indeed be transferred, can be reversed by the addition of normal helper T cells, and is not due to the action of suppressor T cells ( 16). In this experiment we wished to determine whether any differences in anti-TNP antibody avidity existed in irradiated rats reconstituted with carrier-tolerant or normal spleen and lymph node cells. Matched donor groups were left untreated or given 10 mg of SGGSo iv 1 day before sacrifice. Donor spleen and lymph node cells were then removed and washed three times, and 2 x 10s viable cells were transferred iv into syngeneic recipients that had received 900 R of y irradiation 1 hr before transfer. All irradiated rats were maintained on 0.2% Terramycin. One
116
SANFILIPPO
r
AND
SCOTT
‘SC
RESPONSE
o--Q
NORMAL
b-4
CARRIER-TOLERANT
TNP
AT DAY 9
CONCENTRATION
(M)
FIG. 2. Avidity of carrier-tolerant vs control groups in adoptive transfer. Carrier-tolerant or normal spleen plus lymph node cells (2 X 10’ cells) were transferred iv to 900 R, syngeneic recipients and challenged 1 day after cell transfer with TNP-SGG in CFA. Rats receiving carrier-tolerant cells showed a significant reduction in IgG anti-TNP PFC, but the antibody avidity as measured by TNP plaque inhibition with TNP&GG was not affected by carrier tolerance.
day after cell transfer both groups were challenged with 100 pg of TNP-SGG in CFA in each hind footpad, and IgG anti-TNP antibody avidity was determined by plaque inhibition studies 9 days later. As shown in Fig. 2, there was no difference in the anti-TNP antibody avidity between the rats receiving carrier-tolerant or group was 85% normal cells. In this experiment, the control carrier-tolerant unresponsive to TNP. Valence Dependence of Plaque Inhibitors Much of the recent controversy over the use of plaque inhibition as a measure of antibody avidity centers around the extrapolation of plaque inhibition data to determine the affinity or binding constants of the antibody receptor (11). It has been reported (3, 8) that the valence of plaque inhibitor appears to affect the efficiency of inhibition on a per-molar basis of inhibitor. Our findings, using this technique, confirm these data. Inhibition of TNP-PFC by TNP compounds appears to be highly dependent upon the valence of the TNP inhibitor used. Figure 3 shows that as the valence of TNP increases from TNPI-alanine to TNPsSGG to TNPs,,KLH, the efficiency of TNP plaque inhibition on a per-molar basis of TNP increases significantly. Thus, while 1O-p M TNP inhibitor presented as free hapten (TNP-Ala) only inhibits TNP-PFC by lo%, 10e6 M TNP presented as TNPsSGG inhibits TNP-PFC by 60% and the same concentration of TNP used as TNPzOKLH inhibits more than 98%. While this phenomenon is present in both norural and carrier-tolerant rats, it does indicate that the form of presentation of the inhibitor is quite important in reflecting relative avidity. In addition, it appears that the nature of inhibitor conjugate does not itself affect the efficiency of inhibition. Figure 4 compares TNP-PFC inhibition between TNPazRbGG and TNPsa-
HELPER
r
T
CELLS
I$4
AND
ANTIBODY
RESPONSE
AT
AVIDITY
DAY
117
6 . TNP-ALANINE
-NORMAL
TNP
CONCENTRATION
(M)
FK. 3. Valence dependence of TNP plaque inhibitors. The effect of varying the valence of TNP on a macromolecular backbone is shown. As the valence is increased from monovalent TNP to Th’Ps to TNP,, there is an accompanying increase in TNP plaque inhibition per mole of TNP inhibitor. This effect was identical for carrier-tolerant and control groups at day 6 after challenge.
SGG. While RbGG and SGG are similar in size, they are not very cross-reactive antigenically (17). These data show that TNPa2 inhibits to the same extent whether coupIed to RbGG or SGG. Likewise, TNP-plaque inhibition curves obtained using monovalent TNP-alanine or TNP-lysine were identical to each other (data not shown). DISCUSSION The plaque inhibition technique developed by Andersson (7) has proved to be a useful tool in the determination of antibody avidity in recent years. Since its development, however, there has been some controversy over the precise interpreQt.3 0-Q K IOO-
la) TNPJZ
SGG INHIBITOR
PFC AT DAY 6
NORMAL CARRIER
TOLERANT (b) TNPxZ RabGG INHIBITOR
FIG. 4. Effect of molecular backbone of TNP on efficiency of plaque inhibition. Here there is no difference in TNP plaque inhibition between TNP,SGG and TNP,*RbGG. This is true for both carrier-tolerant and control groups. I, points are indicated by arrowheads,
118
SANFILIPPO
AND
SCOTT
tation and correlation of plaque inhibition data with antibody affinity (8, 11). Several studies have been done showing that, on both a theoretical and experimental basis, this technique actually does reflect a measure of antibody avidity (7, 9, 25). It appears less clear whether accurate affinity or binding constants can be determined from the slope and Ijo points of inhibition curves ( I 1) . In our studies, we have confirmed earlier findings that the valence of hapten in the inhibiting compounds may be important in determining the degree of plaque inhibition at a given concentration of hapten. Although we found little change in the slopes of curves obtained using TNP-Ala, TNPsSGG and TNP2,,KLH, we did note a substantial difference in the Ih0 point of these curves when calculated on a per-molar basis of inhibitor. We suggest that the effects of monogamous multivalent binding appear to be a critical factor for this type of analysis (26). That is, a grven amount of free hapten would not be expected to be as competitive in binding antibody as the same amount of hapten in a fixed array on a large molecular backbone. Our incidental finding that the antigenic specificity of this “backbone” plays no major role in affecting plaque inhibition is consistent with this explanation. Thus, while there is some controversy surrounding the validity of binding or affinity constants obtained from such inhibition curves, there is general agreement that plaque inhibition does reflect antibody avidity. It must be pointed out, however, that a recent theoretical analysis of the plaque inhibition assay when multivalent inhibitors are used has suggested that such results may reflect the secretion rate of antibody-forming cells rather than the affinity of antibody secreted (25). While no experimental evidence has yet confirmed this possibility, our findings here must be considered in light of this suggested interpretation of plaque inhibition data. Although the mechanism of carrier-specific T-cell and hapten-specific B-cell collaboration is not yet fully understood, there have been several reports indicating that T-cell populations are involved in determining the avidity of anti-hapten antibody (5, 28). Early work by Siskind et al. (27) showed that the affinity of anti-hapten antibody appears to be related to the nature of the carrier molecule, while T cells were later shown by Gershon and Paul (28) to directly influence the affinity of anti-hapten antibody. This influence on antibody affinity appeared to be related to the total number of T cells transferred to adult, irradiated, thymectomized mice. More recent work by Taniguchi and Tada (5) has expanded these findings. Their work in rabbits showed that thymectomy of adults or treatment with immunosuppressive doses of anti-thymocyte serum resulted in increased affinity of antihapten antibody, supporting the concept that the total number of T cells may have an affect on antibody affinity, Their work showed that such a nonspecific reduction in the total T-cell population resulted in an increase in affinity. In addition, by priming carrier-specific cells before challenge, they observed a relationship between the dose used in carrier priming and the avidity, as well as the total amount of anti-hapten antibody formed. A marked decrease in anti-DNP antibody affinity was seen in rabbits primed with 500 PLg of BGG before challenge with DNP-BGG, although at this priming dose no increase in the total anti-DNP response was noted. At a lower priming dose of 5 pg, however, no effect in anti-DNP affinity appeared but a significant increase occurred in the amount of antibody produced. While this effect may have resulted from the large amount of anti-carrier antibody in circu-
HELPER
T
CELLS
AND
ANTIBODY
AVIDITY
11’1
lation following carrier priming, it is possible that carrier-specific cells were responsible for the observed effect. Katz and others (29, 30) have shown that priming by small doses of carrier results in an increased anti-hapten response upon challenge with hapten-carrier conjugates, but this low-dose priming did not affect the avidity of anti-hapten antibody. The effects of tolerance on antibody affinity have also been studied (31) . Work by Andersson ct al. (32) showed that low-dose tolerance had no apparent effect on antibody avidity whereas high-dose paralysis resulted in lowered avidity. Their work also showed that high dose paralysis affected both T and B cells, while lowdose tolerance was specific for T cells. It is not clear, however, whether this low-dose effect was specific for helper and/or suppressor T cells. Work by Weksler et al. (33) also has shown that high-dose tolerance using DNP-BGG as tolerogen results in a decreased avidity of anti-DNP antibody. However, since the haptenspecific form of the tolerogen is used, this effect is presumably exerted directly at the B-cell level and would not be expected to reflect only T-cell regulation of antihapten antibody avidity. Finally, Taniguchi and Tada (5) have reported that tolerance induced to the carrier alone does appear to affect the affinity of late antihapten antibody. In their study, rabbits were made tolerant to BGG by pretreatment with 500 mg of soluble BGG and then assayed for anti-DNP activity after challenge with DNP-BGG. They found that in some rabbits there was a reduced anti-DNP response along with a significant reduction in the affinity of the anti-DNP antibody produced, while other rabbits in the experiment showed an increased anti-DNP response with no accompanying change in antibody affinity. Although such carrier tolerance would be presumed to act at the helper T-cell level, it is not clear whether the very high dose of soluble BGG given resulted in nonspecific paralysis or true carrier tolerance in the unresponsive rabbits. It is possible that the smaller amount of antibody produced in carrier-tolerant animals compared to controls would be less effective in causing immune elimination of antigen. Thus immune animals would have an additional selective pressure, i.e., antibody, which could drive the system towards higher-affinity antibody production. The results of our studies indicate that helper T cells are not directly involved in determining the avidity of anti-hapten antibodies. Our data show that rats made tolerant to SGG and then challenged with TNP-SGG in CFA give a significantly reduced anti-TNP response compared to nontolerant controls but showed no change in antibody avidity at least during the early maturation of the immune response. JZ’e have previously shown that this carrier-specific tolerance is a direct measure of helper T-cell unresponsiveness, is carrier-specific, and is not a result of suppressor T-cell function or carrier blockage by anti-carrier antibody (16). While we feel that helper T cells do not influence antibody avidity, we have not ruled out effects by suppressor or other T-cell populations. It has been pro1losed that suppressor T cells may act to suppress the proliferation and differentiation of antigen-stimulated B cells (34-37). It is possible that suppressor T cells or another population of T cells may also act to influence antibody avidity. ACKNOWLEDGMENTS
120
SANFILIPPO
AND
SCOTT
REFERENCES 1. Siskind, G., and Benacerraf, B., Advan. Ivnmunol. 10, 1, 1969. 2. Theis, G. A., and Siskind, G. W., J, Zmvnunol. 100, 138, 1968. 3. Davie, J. M., and Paul, W. E., J. Exp. Med. 135, 643, 1972. 4. Andersson, B., J. Exp. Med. 135, 312, 1972. 5. Taniguchi, M., and Tada, T., J. Exp. Med. 139, 108, 1974. 6. Moller, E., Stand. J. Immztnol. 3, 339, 1974. 7. Andersson, B., J. Exp. Med. 132, 77, 1970. 8. Claflin, L., and Merchant, B., Cell. Immunol. 5, 209, 1972. 9. Huchet, R., and Feldmann, M., Eur. J. Immunol. 3, 49, 1973. 10. Davie, J. M., and Paul, W. E., J. Exp. Med. 135, 660, 1972. 11. North, J. R., and Askonis, B. A., Eur. J. Zmmunol. 4, 361, 1974. 12. Green, I., Paul, W. E., and Benacerraf, B., J. Exp. Med. 127, 43, 1968. 13. Henney, C., and Ishizaka, K., J. Zmmunol. 104, 1540, 1970. 14. Segal, S., Globerson, A., and Feldman, M., Cell. Zmrnzozol. 2, 222, 1971. 15. Paul, W. E., Thorbecke, G. J., Siskind, G. W., and Benacerraf, B., Immunology 17, 8.5, 1969. 16. Sanfilippo, F., and Scott, D. W., J. Immunol. 113, 1661, 1974. 17. Scott, D. W., J. Immunol. 112, 1354, 1974. 18. Ornellas, E., Sanfilippo, F., and Scott, D. W., Eur. J. Immzmol. 4, 587, 1974. 19. Sanfilippo, F., and Scott, D. W., submitted for publication. 20. Scott, D. W., J. Immnzmol. 111, 789, 1973. 21. Rittenberg, M., and Pratt, K., Proc. Sot. Exp. Biol. Med. 132, 575, 1969. 22. Lowry, O., Rosebrough, N., Farr, L., and Randall, R., J. Biol. Chem. 193, 265, 1951. 23. Little, J. R., and Eisen, H., In. “Methods in Immunology and Immunochemistry” (C. A. Williams and M. W. Chase, Eds.), Vol. 1, p. 128. Academic Press, New York, 1967. 24. Ornellas, E. P., and Scott, D. W., Cell. Zmmunol. 11, 108, 1974. 2.5. DeLisi, C., and Goldstein, B., Immunochemistry 11, 661, 1974. 26. Hornick, C. L., and Karush, F., In “Topics in Basic Immunology” (M. Sela and M. Prywes, Eds.), pp. 29-36. Academic Press, New York, 1969. 27. Siskind, G., Paul, W. E., and Benacerraf, B., J. Exp. Med. 123, 673, 1966. 28. Gershon, R. K., and Paul, W. E., J. Immuvzol. 106, 872, 1972. 29. Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B., J. Exp, Med. 132, 261, 1970. 30. Ishizaka, K., and Okudaira, H., J. Zmmunol. 110, 1067, 1972. 31. Werblin, T. P., and Siskind, G. W., Transplant. Rev. 8, 104, 1972. 32. Andersson, B., Celada, F., and Asjo, B., Eur. J. Inzmuwol. 4, 788, 1974. 33. Weksler, M. E., Merritts, L. L., Werblin, T. P., and Siskind, G. W., J. Inzmunol. 110, 897, 1973. 34. Gershon, R. K., and Kondo, K., Zmmzlnology 21, 903, 1970. 35. Okumura, K., and Tada, T., J. Zmmunol. 107, 1682, 1971. 36. Tada, T., Okumura, K., and Taniguchi, M., J. Ivnmunol. 108, 1535, 1972. 37. Okumura, K., and Tada, T., Nature New Biol. 245, 180, 1973.
IMMUNOLOGY
21,
112-120 (1976)
The Effects of Carrier-Specific Avidity
in the Anti-Hapten
FRED SANFILIPPO Division
of Immunology,
Helper T-Cell
l AND
Duke Medical Received
DAVID
Tolerance
on Antibody
Response W.
Center, Durham,
SCOTT
North
Carolina,
27710
August 11,1975
Rats rendered tolerant to ultracentrifuged sheep -y-globulin (SGG) have been shown to make a poor anti-trinitrophenyl (TNP)-specific antibody response upon challenge with TNP-SGG in complete Freund’s adjuvant (CFA). We have been able to use this carrier-tolerance system in studying specific helper T-cell unresponsiveness to IgG and IgM antibody responses. By using the plaque inhibition technique to measure antibody avidity, we found that there appears to be no difference in the avidity of antibody responses to TNP between the SGG-tolerant and control groups when both are challenged with TNP-SGG in CFA. This was found to be true in both the 19 and 7s antibody responses ia viva as well as in an adoptive transfer model. In addition, studies on the maturation of 19 and 7s antibody responses showed no differences in antibody avidity between carrier-tolerant and control groups. These findings imply that carrierspecific helper T cells do not play a controlling role in determining whether high- or low-avidity hapten-specific B-cell precursors will proliferate in response to challenge with a hapten-carrier conjugate.
INTRODUCTION The process of clonal selection in the antibody response to specific antigens has been extensively examined in recent years, but the exact nature of how high- vs low-avidity clonal precursor cells are stimulated to proliferate remains a subject of current investigation in many laboratories (l-6). Much of this recent work has focused on the plaque inhibition assay of Andersson (7) as a method of studying the selection process of antibody avidity in response to specific antigen (S-10). This work has provided useful information especially regarding the maturation process of the antibody response, although recent controversy has developed around the validity of the plaque inhibition technique itself (11). In this paper we have attempted to examine the role of the carrier-specific helper T cell 2 in the final determination of antibody avidity in response to specific anti1 Predoctoral Fellow in the Medical Scientist Training Program supported by USPHS Grant No. GM 01678. This publication is also supported in part by USPHS Grant No. A110716. a Abbreviations used: SGG, sheep r-globulin;TNP, trinitrophenyl ; CFA, complete Freund’s adjuvant; T cell, thymus cell ; B cell, bone-marrow-derived cell; PFC, plaque-forming cell; SGGSo, soluble sheep y-globulin; KLH, keyhole limpet hemocyanin; RbGG, rabbit y-globulin; PBS, phosphate-buffered saline ; FCS, fetal calf serum; GRBC, goat erythrocytes ; Ig, immunoglobulin ; DNP, dinitrophenyl ; BGG, bovine y-globulin. 112 Copyright All rights
1976 by Academic Press, oP reproduction in any form
Inc. reserved.
HELPER
T
CELLS
AND
ANTIBODY
113
AVIDITY
gens from the standpoint of carrier-specific helper T-cell tolerance. While it has proven difficult to study selective cell populations in Z&JO, we have been able to study helper T cells by using a carrier-specific tolerance model. By inducing tolerance to a soluble protein, it has been shown that a marked reduction is seen in the subsequent antibody response to a hapten conjugated to the original protein (12-15). Using soluble SGG (SGGSo) as carrier tolerogen and TNP-SGG in CFA as the hapten-carrier conjugate, we have studied this phenomenon in detail, and our findings to date strongly suggest that this system provides a mechanism for studying tolerance of specific helper T cells involved in IgG, IgM and IgE responses (16-19). In this report we have utilized this system to examine the effects of carriertolerant vs normal immune helper T cells on the avidity of hapten-specific antibody responses to hapten+arrier conjugates. Our data suggest that helper T cells are not involved in the final determination of antibody avidity.3 Our incidental findings also confirm recent work by others (S-11) revealing certain ambiguities in the plaque inhibition technique as a method for direct determination of antibody affinity.3 MATERIALS
AND METHODS
A&n&. Eight- to ten-week-old inbred male Lewis rats were purchased from Microbiological Associates (Bethesda, Md.). In each experiment only age- and sex-matched rats were used. Tolerogen. Ultracentrifuged SGGSo was prepared as described previously (20). This tolerogen was prepared separately for each experiment from a stock solution, centrifuged at 100,OOOgat 4°C for 60 min without braking. Only the upper onethird of the solution was used, and a dose of 10 mg in a l-ml volume was injected iv for each rat tolerized. Rats were tolerized 1 day before challenge with haptencarrier complexes. Inznzunogen. Trinitrophenylated sheep y-globulin (TNP-SGG) was prepared as described earlier (17) and emulsified with an equal volume of complete Freund’s adjuvant (Difco) to a final concentration of 1 mg of TNPsSGG/ml in CFA. Tolerant and control rats were challenged with O.l-ml amounts of this immunogen intradermally in both hind footpads for a total dose of 200 g/rat, except where otherwise noted. TNP-conjugated proteins. TNPsSGG, TNP&GG, TNP,,KLH and TNPZ2RbIg were prepared by reacting trinitrobenzene sulfonic acid with the appropriate amount of protein in cacodylate buffer as described previously (17, 21). The final TNP to protein ratios were determined by standard techniques (22, 23) and millimolar dilutions on the basis of total TNP were made in PBS. TNP-p-alanine and r-TNP-L-lysine were purchased from Nutritional Biochemicals, Cleveland, Ohio. Preparation of cell suspensions. Popliteal lymph nodes were removed at various times after challenge and cells were collected in Medium 199 with 570 FCS by gentle agitation through a stainless-steel Susi press (Wm. F. Mayer Co., Yonkers, N.Y.). Cells were washed once and resuspended in Medium 199 with 5% FCS and kept at 4°C until use, usually within 30 min. Plaque inhibition assay of PFC. Popliteal lymph node cells were assayed for plaque-forming cells against TNP-goat red blood cells (TNP-GRBC) which were s Avidity
is defined as the tendency
of an antibody
to form
stable complexes
with
molecular antigen. Affinity is the average binding constant of antibody to antigen.
a macro-
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SANFILIPPO
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prepared by the method of Rittenberg and Pratt (21). The plaque assay used was a modified Jerne plate method as earlier described in detail (24). Indirect (IgG) PFC were developed with a 1: 200 dilution of rabbit anti-rat y-globulin. Inhibition with TNP compounds (TNP-Ala, TNPsSGG, TNPs,,RbGG and TNPssSGG) was carried out by adding the appropriate concentration of inhibitor to the agar overlay tubes. Relative avidity was expressed as the percentage of inhibition of total PFC/rat in each group vs the concentration of TNP inhibitor. Control PFC for each group was the total number of PFC/rat without treatment with inhibitor. For tolerant groups the PFC value was always significantly lower (80%) than control immune groups, and plaque inhibition was calculated as the percentage of reduction from the initial PFC/rat. High-avidity antibody is presumably saturated at a lower concentration of TNP inhibitor than low-avidity antibody, so that low concentrations of the TNP inhibitor block high-avidity antibody from lysing TNP-GRBC (7, 9, 2.5). While avidity measurements using this technique are often expressed only in terms of the Iso point of plaque inhibition, i.e., the concentration of hapten which inhibits the number of PFC by 507 O, we have chosen to present the entire inhibition curve as a measure of avidity. TNP plaque inhibition by this technique was shown to be specific since anti-GRBC PFC were not inhibited by any concentration of any TNP compound. Presentation of data. Groups of three rats were used in each experiment and assays were done in duplicate. Values are expressed as the arithmetic mean per group t standard error. RESULTS Avidity
of Anti-TNP
Antibody
in Carrier
(Helper
T-Cell)-Tolerant
vs Control
Groups
Our previous work in this system has already established various parameters of carrier-specific tolerance (16-19). Briefly, rats pretreated iv with 10 mg of SGGSo and challenged 1 day later with TNP-SGG in CFA show a significant reduction in anti-TNP PFC. This carrier tolerance has been shown to reflect specific unresponsiveness in helper T-cell populations involved in IgM and IgG antibody responses and is apparently not a result of suppressor T-cell activity (16). Figures la and b show that tolerance to SGG does not appear to affect either IgG or IgM anti-TNP antibody avidity in response to, challenge with TNP-SGG. In the first experiment, direct (IgM) PFC (Fig. la) were determined 6 days after challenge with 100 pg of TNP-SGG in CFA. At this time and with this challenge dose, the IgM anti-TNP response in the control carrier-tolerant group is 16% of the control nontolerant group, i.e., 84% tolerant. Both groups, however, show an identical percentage of reduction in total PFC/rat upon plaque inhibition with 1O-8-1@* M TNP. Figure lb shows that the avidity of the IgG response at day 20 after challenge with 3 pg of TNP-SGG is also unaffected by carrier tolerance. In this second experiment the control carrier-tolerant group showed a 94% decrease in anti-TNP PFC as compared to the nontolerant control group. In both experiments TNPsSGG was used as PFC inhibitor. To determine whether the avidity of anti-hapten antibody was affected by carrier tolerance as the IgG response matured, these experiments were repeated at days 20 and 29 after challenge. In addition, challenge doses were varied from 3 to 100
HELPER
I
T
CELLS
AND
b-4
ANTIBODY
NORMAL CARRIER
TOLERANT (b)
TNP
11.5
AVIDITY
CONCENTRATION
IyG
PFC AT DAY 20
(M)
FIG. 1. Effect of carrier-specific (helper T-cell) tolerance on anti-hapten antibody avidity. Carrier-tolerant groups were pretreated with 10 mg of SGGSo 1 day prior to challenge with TNP-SGG in CFA. Anti-TNP antibody avidity is expressed as percentage of TNP PFC inhibition vs concentration of TNP as TNPSGG inhibitor, with 1, points indicated by arrowhead. (a), Comparison of IgM anti-TNP plaque inhibition of carrier-tolerant and control groups at day 6 after challenge. (b), Comparison of IgG anti-TNP avidity of carrier-tolerant and control groups at day 20. In both experiments, anti-TNP antibody was significantly reduced in tolerant groups, but, as the graphs show, there were no apparent differences in avidity.
pg of TNP-SGG, and in all cases there were no significant differences in avidity between the carrier-tolerant and nontolerant groups. Preliminary findings also indicate that the avidity of anti-TNP antibody in the secondary response is also unaffected by carrier tolerance but that rats made hapten (B cell) tolerant by pretreatment with TNP-Rat Ig showed a significant reduction in anti-TNP avidity (data not shown). Avidity
of Carrier-Tolerant
vs Control Groups in Adoptive
Transfer
In order to rule out the possibility that carrier-specific tolerance in our in vivo system is merely due to an ongoing competition between tolerogen and immunogen or that the carrier determinants of the hapten-carrier immunogen are blocked by undetectable levels of anti-carrier antibodies responding to the tolerogen, we have carried out several studies using an adoptive transfer model. Briefly, normal donor rats or rats made tolerant to carrier protein 1 day before transfer are sacrificed, their spleen and lymph node cells pooled in separate groups and, after thorough washing, cells are transferred iv into lethally irradiated syngeneic recipients. Using this adoptive transfer model, we have been able to show that such carrier tolerance can indeed be transferred, can be reversed by the addition of normal helper T cells, and is not due to the action of suppressor T cells ( 16). In this experiment we wished to determine whether any differences in anti-TNP antibody avidity existed in irradiated rats reconstituted with carrier-tolerant or normal spleen and lymph node cells. Matched donor groups were left untreated or given 10 mg of SGGSo iv 1 day before sacrifice. Donor spleen and lymph node cells were then removed and washed three times, and 2 x 10s viable cells were transferred iv into syngeneic recipients that had received 900 R of y irradiation 1 hr before transfer. All irradiated rats were maintained on 0.2% Terramycin. One
116
SANFILIPPO
r
AND
SCOTT
‘SC
RESPONSE
o--Q
NORMAL
b-4
CARRIER-TOLERANT
TNP
AT DAY 9
CONCENTRATION
(M)
FIG. 2. Avidity of carrier-tolerant vs control groups in adoptive transfer. Carrier-tolerant or normal spleen plus lymph node cells (2 X 10’ cells) were transferred iv to 900 R, syngeneic recipients and challenged 1 day after cell transfer with TNP-SGG in CFA. Rats receiving carrier-tolerant cells showed a significant reduction in IgG anti-TNP PFC, but the antibody avidity as measured by TNP plaque inhibition with TNP&GG was not affected by carrier tolerance.
day after cell transfer both groups were challenged with 100 pg of TNP-SGG in CFA in each hind footpad, and IgG anti-TNP antibody avidity was determined by plaque inhibition studies 9 days later. As shown in Fig. 2, there was no difference in the anti-TNP antibody avidity between the rats receiving carrier-tolerant or group was 85% normal cells. In this experiment, the control carrier-tolerant unresponsive to TNP. Valence Dependence of Plaque Inhibitors Much of the recent controversy over the use of plaque inhibition as a measure of antibody avidity centers around the extrapolation of plaque inhibition data to determine the affinity or binding constants of the antibody receptor (11). It has been reported (3, 8) that the valence of plaque inhibitor appears to affect the efficiency of inhibition on a per-molar basis of inhibitor. Our findings, using this technique, confirm these data. Inhibition of TNP-PFC by TNP compounds appears to be highly dependent upon the valence of the TNP inhibitor used. Figure 3 shows that as the valence of TNP increases from TNPI-alanine to TNPsSGG to TNPs,,KLH, the efficiency of TNP plaque inhibition on a per-molar basis of TNP increases significantly. Thus, while 1O-p M TNP inhibitor presented as free hapten (TNP-Ala) only inhibits TNP-PFC by lo%, 10e6 M TNP presented as TNPsSGG inhibits TNP-PFC by 60% and the same concentration of TNP used as TNPzOKLH inhibits more than 98%. While this phenomenon is present in both norural and carrier-tolerant rats, it does indicate that the form of presentation of the inhibitor is quite important in reflecting relative avidity. In addition, it appears that the nature of inhibitor conjugate does not itself affect the efficiency of inhibition. Figure 4 compares TNP-PFC inhibition between TNPazRbGG and TNPsa-
HELPER
r
T
CELLS
I$4
AND
ANTIBODY
RESPONSE
AT
AVIDITY
DAY
117
6 . TNP-ALANINE
-NORMAL
TNP
CONCENTRATION
(M)
FK. 3. Valence dependence of TNP plaque inhibitors. The effect of varying the valence of TNP on a macromolecular backbone is shown. As the valence is increased from monovalent TNP to Th’Ps to TNP,, there is an accompanying increase in TNP plaque inhibition per mole of TNP inhibitor. This effect was identical for carrier-tolerant and control groups at day 6 after challenge.
SGG. While RbGG and SGG are similar in size, they are not very cross-reactive antigenically (17). These data show that TNPa2 inhibits to the same extent whether coupIed to RbGG or SGG. Likewise, TNP-plaque inhibition curves obtained using monovalent TNP-alanine or TNP-lysine were identical to each other (data not shown). DISCUSSION The plaque inhibition technique developed by Andersson (7) has proved to be a useful tool in the determination of antibody avidity in recent years. Since its development, however, there has been some controversy over the precise interpreQt.3 0-Q K IOO-
la) TNPJZ
SGG INHIBITOR
PFC AT DAY 6
NORMAL CARRIER
TOLERANT (b) TNPxZ RabGG INHIBITOR
FIG. 4. Effect of molecular backbone of TNP on efficiency of plaque inhibition. Here there is no difference in TNP plaque inhibition between TNP,SGG and TNP,*RbGG. This is true for both carrier-tolerant and control groups. I, points are indicated by arrowheads,
118
SANFILIPPO
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tation and correlation of plaque inhibition data with antibody affinity (8, 11). Several studies have been done showing that, on both a theoretical and experimental basis, this technique actually does reflect a measure of antibody avidity (7, 9, 25). It appears less clear whether accurate affinity or binding constants can be determined from the slope and Ijo points of inhibition curves ( I 1) . In our studies, we have confirmed earlier findings that the valence of hapten in the inhibiting compounds may be important in determining the degree of plaque inhibition at a given concentration of hapten. Although we found little change in the slopes of curves obtained using TNP-Ala, TNPsSGG and TNP2,,KLH, we did note a substantial difference in the Ih0 point of these curves when calculated on a per-molar basis of inhibitor. We suggest that the effects of monogamous multivalent binding appear to be a critical factor for this type of analysis (26). That is, a grven amount of free hapten would not be expected to be as competitive in binding antibody as the same amount of hapten in a fixed array on a large molecular backbone. Our incidental finding that the antigenic specificity of this “backbone” plays no major role in affecting plaque inhibition is consistent with this explanation. Thus, while there is some controversy surrounding the validity of binding or affinity constants obtained from such inhibition curves, there is general agreement that plaque inhibition does reflect antibody avidity. It must be pointed out, however, that a recent theoretical analysis of the plaque inhibition assay when multivalent inhibitors are used has suggested that such results may reflect the secretion rate of antibody-forming cells rather than the affinity of antibody secreted (25). While no experimental evidence has yet confirmed this possibility, our findings here must be considered in light of this suggested interpretation of plaque inhibition data. Although the mechanism of carrier-specific T-cell and hapten-specific B-cell collaboration is not yet fully understood, there have been several reports indicating that T-cell populations are involved in determining the avidity of anti-hapten antibody (5, 28). Early work by Siskind et al. (27) showed that the affinity of anti-hapten antibody appears to be related to the nature of the carrier molecule, while T cells were later shown by Gershon and Paul (28) to directly influence the affinity of anti-hapten antibody. This influence on antibody affinity appeared to be related to the total number of T cells transferred to adult, irradiated, thymectomized mice. More recent work by Taniguchi and Tada (5) has expanded these findings. Their work in rabbits showed that thymectomy of adults or treatment with immunosuppressive doses of anti-thymocyte serum resulted in increased affinity of antihapten antibody, supporting the concept that the total number of T cells may have an affect on antibody affinity, Their work showed that such a nonspecific reduction in the total T-cell population resulted in an increase in affinity. In addition, by priming carrier-specific cells before challenge, they observed a relationship between the dose used in carrier priming and the avidity, as well as the total amount of anti-hapten antibody formed. A marked decrease in anti-DNP antibody affinity was seen in rabbits primed with 500 PLg of BGG before challenge with DNP-BGG, although at this priming dose no increase in the total anti-DNP response was noted. At a lower priming dose of 5 pg, however, no effect in anti-DNP affinity appeared but a significant increase occurred in the amount of antibody produced. While this effect may have resulted from the large amount of anti-carrier antibody in circu-
HELPER
T
CELLS
AND
ANTIBODY
AVIDITY
11’1
lation following carrier priming, it is possible that carrier-specific cells were responsible for the observed effect. Katz and others (29, 30) have shown that priming by small doses of carrier results in an increased anti-hapten response upon challenge with hapten-carrier conjugates, but this low-dose priming did not affect the avidity of anti-hapten antibody. The effects of tolerance on antibody affinity have also been studied (31) . Work by Andersson ct al. (32) showed that low-dose tolerance had no apparent effect on antibody avidity whereas high-dose paralysis resulted in lowered avidity. Their work also showed that high dose paralysis affected both T and B cells, while lowdose tolerance was specific for T cells. It is not clear, however, whether this low-dose effect was specific for helper and/or suppressor T cells. Work by Weksler et al. (33) also has shown that high-dose tolerance using DNP-BGG as tolerogen results in a decreased avidity of anti-DNP antibody. However, since the haptenspecific form of the tolerogen is used, this effect is presumably exerted directly at the B-cell level and would not be expected to reflect only T-cell regulation of antihapten antibody avidity. Finally, Taniguchi and Tada (5) have reported that tolerance induced to the carrier alone does appear to affect the affinity of late antihapten antibody. In their study, rabbits were made tolerant to BGG by pretreatment with 500 mg of soluble BGG and then assayed for anti-DNP activity after challenge with DNP-BGG. They found that in some rabbits there was a reduced anti-DNP response along with a significant reduction in the affinity of the anti-DNP antibody produced, while other rabbits in the experiment showed an increased anti-DNP response with no accompanying change in antibody affinity. Although such carrier tolerance would be presumed to act at the helper T-cell level, it is not clear whether the very high dose of soluble BGG given resulted in nonspecific paralysis or true carrier tolerance in the unresponsive rabbits. It is possible that the smaller amount of antibody produced in carrier-tolerant animals compared to controls would be less effective in causing immune elimination of antigen. Thus immune animals would have an additional selective pressure, i.e., antibody, which could drive the system towards higher-affinity antibody production. The results of our studies indicate that helper T cells are not directly involved in determining the avidity of anti-hapten antibodies. Our data show that rats made tolerant to SGG and then challenged with TNP-SGG in CFA give a significantly reduced anti-TNP response compared to nontolerant controls but showed no change in antibody avidity at least during the early maturation of the immune response. JZ’e have previously shown that this carrier-specific tolerance is a direct measure of helper T-cell unresponsiveness, is carrier-specific, and is not a result of suppressor T-cell function or carrier blockage by anti-carrier antibody (16). While we feel that helper T cells do not influence antibody avidity, we have not ruled out effects by suppressor or other T-cell populations. It has been pro1losed that suppressor T cells may act to suppress the proliferation and differentiation of antigen-stimulated B cells (34-37). It is possible that suppressor T cells or another population of T cells may also act to influence antibody avidity. ACKNOWLEDGMENTS
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REFERENCES 1. Siskind, G., and Benacerraf, B., Advan. Ivnmunol. 10, 1, 1969. 2. Theis, G. A., and Siskind, G. W., J, Zmvnunol. 100, 138, 1968. 3. Davie, J. M., and Paul, W. E., J. Exp. Med. 135, 643, 1972. 4. Andersson, B., J. Exp. Med. 135, 312, 1972. 5. Taniguchi, M., and Tada, T., J. Exp. Med. 139, 108, 1974. 6. Moller, E., Stand. J. Immztnol. 3, 339, 1974. 7. Andersson, B., J. Exp. Med. 132, 77, 1970. 8. Claflin, L., and Merchant, B., Cell. Immunol. 5, 209, 1972. 9. Huchet, R., and Feldmann, M., Eur. J. Immunol. 3, 49, 1973. 10. Davie, J. M., and Paul, W. E., J. Exp. Med. 135, 660, 1972. 11. North, J. R., and Askonis, B. A., Eur. J. Zmmunol. 4, 361, 1974. 12. Green, I., Paul, W. E., and Benacerraf, B., J. Exp. Med. 127, 43, 1968. 13. Henney, C., and Ishizaka, K., J. Zmmunol. 104, 1540, 1970. 14. Segal, S., Globerson, A., and Feldman, M., Cell. Zmrnzozol. 2, 222, 1971. 15. Paul, W. E., Thorbecke, G. J., Siskind, G. W., and Benacerraf, B., Immunology 17, 8.5, 1969. 16. Sanfilippo, F., and Scott, D. W., J. Immunol. 113, 1661, 1974. 17. Scott, D. W., J. Immunol. 112, 1354, 1974. 18. Ornellas, E., Sanfilippo, F., and Scott, D. W., Eur. J. Immzmol. 4, 587, 1974. 19. Sanfilippo, F., and Scott, D. W., submitted for publication. 20. Scott, D. W., J. Immnzmol. 111, 789, 1973. 21. Rittenberg, M., and Pratt, K., Proc. Sot. Exp. Biol. Med. 132, 575, 1969. 22. Lowry, O., Rosebrough, N., Farr, L., and Randall, R., J. Biol. Chem. 193, 265, 1951. 23. Little, J. R., and Eisen, H., In. “Methods in Immunology and Immunochemistry” (C. A. Williams and M. W. Chase, Eds.), Vol. 1, p. 128. Academic Press, New York, 1967. 24. Ornellas, E. P., and Scott, D. W., Cell. Zmmunol. 11, 108, 1974. 2.5. DeLisi, C., and Goldstein, B., Immunochemistry 11, 661, 1974. 26. Hornick, C. L., and Karush, F., In “Topics in Basic Immunology” (M. Sela and M. Prywes, Eds.), pp. 29-36. Academic Press, New York, 1969. 27. Siskind, G., Paul, W. E., and Benacerraf, B., J. Exp. Med. 123, 673, 1966. 28. Gershon, R. K., and Paul, W. E., J. Immuvzol. 106, 872, 1972. 29. Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B., J. Exp, Med. 132, 261, 1970. 30. Ishizaka, K., and Okudaira, H., J. Zmmunol. 110, 1067, 1972. 31. Werblin, T. P., and Siskind, G. W., Transplant. Rev. 8, 104, 1972. 32. Andersson, B., Celada, F., and Asjo, B., Eur. J. Inzmuwol. 4, 788, 1974. 33. Weksler, M. E., Merritts, L. L., Werblin, T. P., and Siskind, G. W., J. Inzmunol. 110, 897, 1973. 34. Gershon, R. K., and Kondo, K., Zmmzlnology 21, 903, 1970. 35. Okumura, K., and Tada, T., J. Zmmunol. 107, 1682, 1971. 36. Tada, T., Okumura, K., and Taniguchi, M., J. Ivnmunol. 108, 1535, 1972. 37. Okumura, K., and Tada, T., Nature New Biol. 245, 180, 1973.
