Immunology 1977 33 373

Effects of whole-body irradiation

on

antibody affinity*

G. GORINI, L. ADORINI, DIANA BORASCHI, A. DI MICHELE & G. DORIA CNEN-Euratom Immunogenetics Group, Laboratory of Radiopathology, C.S.N. Casaccia

(Rome), Italy

Received 9 December 1976;

accepted for publication

Summary. Mice exposed to a sublethal dose of Xrays were immunized with alum-precipitated DNPKLH (dinitrophenyl-keyhole limpet haemocyanin) and B. pertussis either before or after irradiation. The primary anti-DNP antibody response was evaluated during 8 weeks after immunization by the equilibrium dialysis technique using ammonium sulphate- precipitated serum globulins and the ligand 3H-labelled e-DNP-L-Lysine. The serum concentrations of antibody sites in mice immunized 1-5 days before or 2 h-8 weeks after 450 rad were below the values in unirradiated controls at all bleeding times. Antibody affinity, however, was found to be up to 20 fold higher in irradiated mice than in control mice when antigen was injected before, or 3-8 weeks after, irradiation. Spleen cells from mice exposed to 450 rad 1-9 weeks before killing were stimulated in vitro with PHA, ConA, or LPS. Recovery profiles of mitotic responsiveness suggest that enhancement of antibody affinity in irradiated mice could result from relative lack of suppressor T cells.

INTRODUCTION The depressive effect of whole body X-irradiation on the antibody response has been extensively investigated. Maximal depression of all parameters of the

13

January 1977

immune response is observed when antigen is injected shortly before or immediately after irradiation. Recovery of the immune response in animals immunized after sublethal irradiation starts at 1 week and may be complete 2 months after radiation exposure. If animals are immunized a few or several days before irradiation some parameters of the antibody response are unaffected while others may display lower or higher values than normal, depending on several factors including the nature and physical form of the antigen (Taliaferro, Taliaferro & Jaroslow, 1964). It has been demonstrated that in sublethally irradiated mice primed with DNP-KLH (Dinitrophenyl-Keyhole Limpet Haemocyanin) after irradiation the anti-DNP secondary response is characterized by antibody affinity greater than that in unirradiated controls (Doria, Gorini & Di Michele, 1977). The present study was undertaken to investigate antibody affinity during the primary response in mice immunized before or after a sublethal dose of X-rays. Radiation induced changes of lymphocyte populations were evaluated at the time of immunization by the mitotic response to PHA, ConA, and LPS of spleen cells in vitro. MATERIALS AND METHODS Mice

* Supported by CNEN-Euratom Association Contract. Publication No. 1402 of the Euratom Biology Division. Correspondence: Dr G. Gorini, CNEN-Euratom Immunogenetics Group, Laboratory of Radiopathology, C.S.N. Casaccia (Rome), Italy.

Inbred C3HeB/FeJ female mice were caged in groups of 8 and allowed free access to pellets of standard food and chlorinated water. All experiments were performed with 3-4-month-old mice. 373

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Irradiation Mice were exposed to a total-body X-ray dose of 450 rad. Irradiation conditions have been described previously (Doria & Agarossi, 1968).

Log (R/(N-R)) = a Log Ko + a Log C. From linear regression analysis Ko was calculated and expressed in L/M as the value of 1/C at which 50 per cent of the total antibody sites are occupied.

Immunization Mice were immunized with a single intraperitoneal injection of 0-2 ml PBS (phosphate buffered saline, pH 7-2) containing 0-1 mg alum-precipitated DNPKLH (62 DNP groups/KLH molecule), purchased from Calbiochem, and I x 109 killed Bordetella pertussis (Sclavo, Italy). Groups of 40 mice were immunized 1 or 5 days before, or 2 h, 2, 3, 4 or 8 weeks after irradiation. Eight mice ofeach group were bled by decapitation 1, 2, 4, 6 and 8 weeks after immunization. At each bleeding time, sera from mice of the same group were pooled and stored at - 200. The same schedule of immunization and bleeding was applied to control groups of 40 unirradiated mice.

In vitro mitotic response Groups of 4 mice were exposed to a single dose of X-rays (450 rad) at different times and were all killed on the same day. The time intervals between irradiation and killing were: 2 h, 1, 2, 3, 4, 5, 6, 7, 8 and 9 weeks. Spleen cells from 4 irradiated mice or 4 unirradiated controls were pooled and stimulated in vitro with PHA, ConA, and LPS in separate cultures, as previously described (Adorini, Gorini, Covelli, Ballardin, Di Michele, Bassani, Metalli & Doria, 1976). Spleen cells from mice of the same group were cultured in RPMI 1640 medium (Eurobio) supplemented with I % penicillin-streptomycin mixture (Microbiological Associates), 1% L-Glutamine (Microbiol. Associates), 10% fetal calf serum (Rehatuin, Reheis), 2% HEPES (Eurobio). Aliquots of 0 1 ml of each cell suspension (6 x 106 nucleated spleen cells/ml) were distributed in wells of microtest tissue culture plates (n. 3040, Falcon Plastics) and cultured in triplicate together with 0 1 ml of medium alone, or containing a mitogen at 7 concentrations: PHA (Wellcome) from 1 to 100 ,g/ml, ConA (Calbiochem) from 1 to 100 pg/ml, LPS (from E. Coli 055 :B5, Difco) from 1 to 500 ug/ml. After 48 h of culture at 370 in humid air with 5% CO2, 05 ,uCi of methyl-[3H]-thymidine, specific activity 2 Ci/ mmol (Amersham) in 10 ,pl PBS were added to each well. Sixteen hours later, the cells were harvested from wells and transferred on to glass fibre paper filters by a multiple cell culture harvester (Skatron). The filters were dried and placed in plastic scintillation vials (Packard) together with 5 ml of Scintillation fluid (Permafluor, Packard) and counted in a Packard Tri-Carb scintillation spectrometer (counting efficiency about 20%). The maximal response of spleen cells from both irradiated and control mice was found at a final concentration of 5 pg/ml for PHA, 5 ug/ml for ConA, and 50 pg/ml for LPS. Results are expressed as arithmetic mean of cpm from triplicate cultures ± standard error (s.e.) or as per cent of the cpm response of normal spleen cells.

Equilibrium dialysis The technique used to determine the serum concenration of total antibody sites and antibody affinity for DNP-lysine was basically that of Werblin & Siskind (1972) and has been described in detail elsewhere (Doria et al., 1977). Briefly, equilibrium dialysis was carried out at 40 using globulin samples prepared by precipitation of each serum pool with 50% ammonium sulphate, using 3H-labelled (e-DNPL-Lysine (New England Nuclear) as ligand at concentrations ranging from about 2-5 x 10-6 to 1 x 10- 8 moles/litre in PBS. Values of R (concentration of bound antibody sites) and C (concentration of free ligand) obtained at the 4-6 higher consecutive ligand concentrations were used to calculate the concentration of total (bound and free) antibody sites (N) for DNP-lysine in a sample. The binding data plotted as 1 /R versus 1/C were fitted by a straight line. The reciprocal of the intercept calculated by linear regression analysis and expressed in moles/litre provided the value of N in the globulin sample and, taking into account the dilutions involved from the preparation to the use of the globulin sample, in the original serum. Antibody affinity for DNP-lysine was defined as the average association constant (Ko) and calculated after plotting the binding data as Log (R/(N-R)) versus Log C. Only binding data falling within 30-70 per cent of total antibody sites were fitted by a straight linc according to the Sips equation:

RESULTS No mortality was observed in irradiated mice or unirradiated controls.

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antigen was injected 1 day before 450 rad, antibody affinity of irradiated mice was initially lower than in unirradiated animals, but it increased at a very fast rate and displayed at 4, 6 and 8 weeks after immunization a similar enhancement to that observed when mice were immunized 5 days before irradiation. When mice were immunized 2 h or 2 weeks after 450 rad antibody affinity was always below the control values, although it increased with time after immunization. Antigen injection at 3, 4 or 8 weeks after irradiation yielded antibodies of greater average affinity in irradiated than in control mice. For-each interval between irradiation and immunization antibody affinity was initially lower than in unirradiated animals, then rose at a very high rate to attain levels well above the controls. As the interval between irradiation and subsequent immunization was prolonged, the difference in affinity increased sharply and then decreased to a lower, but appreciable, level. A maximal difference of 20 fold was observed when mice were immunized 4 weeks after irradiation and bled 8 weeks after antigen injection.

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Figure 1. Binding data from equilibrium dialysis of one immunoglobulin preparation reacted against various concentrations of DNP-lysine for determination of the concentration (N) (a) and affinity (Ko) (b) of antibody sites. See the Materials and methods section for symbols. The data falling within 30-70% of total antibody sites are included within the dotted lines.

Antibody affinity Fig. gives an example of binding data obtained by the equilibrium dialysis technique as used in the present study. Fig. 2 (upper part) shows the results of two experiments illustrating the effect of irradiation on antibody affinity. In one experiment, antigen was injected before irradiation; in the other one, alter irradiation. The latter experiment was repeated and provided similar data. Good reproducibility of the results is also apparent in Fig. 2 from comparison of affinity values of the unirradiated controls in the two experiments. When antigen was injected 5 days before 450 rad, irradiation brought about a consistent enhancement of antibody affinity. From the first bleeding on, antibody affinity of irradiated mice increased at a faster rate than in non-irradiated animals, reached a peak at 6 weeks after immunization, then declined to a value still above the level in control animals. When

Antibody concentration The data shown in the lower part of Fig. 2 represent the serum concentration of total antibody sites for DNP-lysine. The depressive effect of irradiation was very profound when mice were immunized either before or after exposure to 450 rad. Maximum depression was observed when antigen was injected 2 h after irradiation, as stressed by the fact that antibodies could not be detected in the serum of mice killed and 2 weeks after immunization. The profound depression persisted for 2 weeks after irradiation. At longer intervals between irradiation and immunization the depressive effect was less pronounced, indicating that recovery of the immune response did occur but was not yet complete at 8 weeks postirradiation. Radiation damage of the antibody response was also apparent when mice were immunized 1 or 5 days before exposure to 450 rad. In vitro mitotic response Table shows the mitotic response to PHA, ConA, and LPS of spleen cells from mice unirradiated or irradiated at various times before culture. The results from two experiments are reported as mean c.p.m. from triplicate cultures. The results of stimulated

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G. Gorini et al.

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Figure 2. Effects of irradiation on serum antibody affinity and concentration of mice immunized with DNP-KLH. Affinity (upper part) and serum concentration (lower part) of antibody sites for DNP-lysine in irradiated (- -) and unirradiated ( mice. The results are from two experiments: immunization 1-5 days before 450 rad; immunization 2 h-8 weeks after 450 rad. The same control ( ) is shown for each experiment. Intervals between irradiation and immunization are as follows: (a) 5 days; (b) 1 day; (c) 2 hr; (d) 2 weeks; (e) 3 weeks; (f) 4 weeks; (g) 8 weeks. Columns I and II = antigen before 450 rad; columns II-VII = antigen after 450 rad. -

cultures from the same two experiments were pooled and the responses of cells from irradiated mice were expressed as per cent of unirradiated controls (Fig. 3). These data show that the responses to PHA, ConA, and LPS were very depressed immediately after irradiation. Rapid recovery of the responses to PHA and ConA started 4 weeks after irradiation and reached about 60% of the control values three weeks later without further increase during the subsequent weeks. The recovery of the mitotic response to LPS started 1 week after irradiation, attained about 80% of the control value at the 6th week and then slightly declined.

DISCUSSION The main finding of the present work is that a sublethal dose of X-ray can affect the primary immune response to DNP-KLH, in terms not only of serum

concentration but also of average affinity of haptenspecific antibodies. The serum concentration of antibody sites for DNP-lysine was found depressed regardless of whether antigen was injected before or after irradiation, a finding consistent with the literature (Taliaferro et al., 1964). Antibody affinity was found to be higher in irradiated than in control mice when antigen was injected before exposure to 450 rad. The irradiation could possibly lead to alterations of the population of immunologically competent cells favouring the selection by antigen of B cells with high affinity receptors (Siskind & Benacerraf, 1969). Since the B cell population can produce antibodies of higher or lower affinity upon interaction with helper (Gershon & Paul, 1971) or suppressor (Tada, Taniguchi & Takemori, 1975) T cells, respectively, a relative lack of suppressor T cells could explain the increase of affinity. This possibility is supported by the observa-

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