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Analysis of the temporal changes in the avidity of primary IgM and IgG antibody at the cellular level
CELLULAR
IMMUNOLOGY
Analysis
205-211
11,
(1974)
of the Temporal Changes in the Avidity of Primary IgM and IgG Antibody at the Cellular Level T. L. ROSZMAN
Department
of
Cell
Biology, College of Medicine, Lexington, Kentucky 4506 Received
Jwne
University
of Kentucky,
29, 1973
The avidity of primary IgM and IgG anti-bovine serum albumin antibody was measured at the cellular level. The avidity of IgG antibody secreted by plaqueforming cells increased with time as evidenced by the fact that progressively less bovine serum albumin was required to inhibit 50’76 of the plaque-forming cells. Analysis of various avidity subpopulations of IgG plaque-forming cells, furthermore, demonstrated that there was a temporal attrition of low avidity IgG plaque-forming cells resulting in the dominance of high avidity IgG plaque-forming cells. Thus,
maturation of IgG antibody was occurring during the primary antibody response to bovine serum albumin. Similar analyses of the IgM antibody secreted by plaqueforming cells indicated that the avidity the exception of a marked increase
subpopulations ing
that
remained
constant
throughout
the response
with
on Day 8. Heterogeneity of the various avidity of IgRI plaque-forming cells was a hallmark of the response indicat-
maturation
is not
a major
feature
of the IghI
response.
IXTRODUCTION It is well known that a temporal increase in the antigen binding characteristics of IgG antibody occurs after primary immunization as a result of antigen preferentially selecting those precursors of the antibody-forming cells with higher affinity receptors (1). Maturation of the IgG response has been observed at the humoral (24) as well as the cellular level (5, 6). Andersson (7) has demonstrated, employing the plaque inhibition assay, that the time-related increases in the avidity of IgG antibody secreted by plaque-forming cells (PFC) is similar to that observed in the serum. A correlation, furthermore, was found to exist between the rate of increase in the avidity of IgG antibody at the serum and cellular level with the increase
avidity
of antigen-binding
cells during
the immune
response
to dinitro-
phenol-guinea pig albumin (8). Similar studies concerned with determining whether or not the IgM responses undergoes antigen-driven maturation have yielded conflicting results (9-15). Recently, however, Claflin et al. (16j have observed that the avidity of primary IgM antibody produced by PFC does increase with time and that the maturation process is complete by 9 to 10 days after primary immunization. In the present study, the temporal changes in the avidity of IgM and IgG antibody secreted by PFC during the primary antibody response to bovine serum albumin (BSA) was examined employing a plaque inhibition assay. The results 205 Copyri ht Q 1974 by Academic Press, All rig e;ts of reproduction in any form
Inc. reserved.
206
ROSZMAN
are consistent with the conclusion that a maturation process is operative as evidenced by the fact that the avidity of IgG antibody does increase with time. The kinetics of development of the avidity of IgM antibody differed considerably from ‘that of IgG antibody which indicates that antigen-driven maturation is not a feature of the IgM primary antibody response. MATERIALS
AND
METHODS
Animals and immunization. Female New Zealand white rabbits 3-6 months of age were immunized with 0.5 mg of BSA (Miles Laboratories, Inc., Kankakee, IL) incorporated into complete Freund’s adjuvant (Difco Labs, Detroit, MI) in each hind footpad. Rabbits were sacrificed at various times after primary immunization and popliteal lymph nodes removed. Preparation of cell suspensions. Cell suspensions were prepared from the spleens and popliteal lymph nodes according to the method of Roszman et al. (17). The final concentration of the nucleated cells was adjusted to l-2 x log/ml in Eagle’s minimum essential medium (MEM, Grand Island Biological Co., Grand Island, NY). Preparation of sensitized sheep red blood cells. The carbodiimide method of Gunn and Roszman (14) was employed to couple BSA to sheep red blood cells (SRBC). Cells were coupled in the presence of 100 mg/ml of BSA since preliminary experiments demonstrated that maximal numbers of both IgM and IgG PFC were detected when this concentration of protein was used for coupling. The sensitized SRBC were resuspended to a final concentration of 7% (v/v) in MEM. Plaque assay technique. A modification of the Cunningham and Szenberg liquid plaque assay (18) was employed. Two siliconized microscope slides were taped together with double side tape (Dubl-Stik Tape, Kleen-Stik Prods., Newark, NJ) to form two chambers with a volume of approximately 0.05 ml each. Immunoglobulin G AFC were developed by facilitation with a goat anti-rabbit IgG serum (Cappel Laboratory, Downingtown, PA) at a final dilution of 1 : 50. This facilitating antiserum caused complete inhibition of IgM AFC. The plaquing chambers were incubated at 37°C for 30-60 min to develop IgM AFC and 90-120 min for IgG AFC. After incubation, the plaques were counted under 35 X magnification. In addition to developing more rapidly, IgM PFC were completely inhibited by concanavalin A (19)) whereas IgG PFC were not. Plaque inhibition assay technique. The method of Andersson (7) was employed to determine the percent inhibition of IgM and IgG PFC by various concentrations of free BSA. A mixture containing guinea pig complement, BSA sensitized SRBC, facilitating antiserum when needed, and an aliquot of lymphoid cells containing between 400 and 800 PFC/O.l ml was prepared. A 0.1 ml aliquot of this was added to each of a series of tubes containing 0.1 ml of increasing concentraItions of BSA in MEM ranging from 0.1 to 100 pg. Duplicate chambers were filled with these mixtures and the number of PFC determined as previously described. That concentration of BSA which inhibited 50% of PFC was taken as the avidity (ho). Analysis of avidity subpopula’tions. The method of Davies and Paul (8) was employed to determine the absolute number of IgM and IgG PFC in each avidity subgroup.
AVIDITY
01’
Days after immunization
PRIMARY
IGM
AND
Number
IGG
of I’FC,‘lV
AS7‘1
HOI)\
nucleated
cells
1gn1
.i
212 f 108.1 f
6
i x 0 10 11
132” 115
41 It22
935
zk 190
974 1725
185
r!z 36
1166
113 39
f
f- 141 108.3 * 506 2% f 102
16
l
16
.X3 rt
16
f f
257 .362
i
230
676
RESULTS Kinetics of the IgM and IgG pviuzary antibody respomc. The appearance of IgM and IgG PFC in the popliteal lymph nodes at various times after a single injection of antigen is illustrated in Table 1. The results represent the mean of j-7 separate experiments. There is a rapid rise and decline of IgM and IgG PFC with maximum numbers of IgG PFC appearing one day after the IgM peak. Average avidity of IgM and IgG atztibody secreted by PFC. Inhibition experiments were performed in order to determine the temporal change in the avidity of IgM and IgG antibody synthesized during the primary antibody response. The data from three representative experiments showing inhibition of the IgM I’FC are presented in Fig. 1. The highest avidity IgM antibody was produced on Day S of the response with an 150value of 4.5 pg BSA as compared to Ir,o values of 15.1 and 11.5 ,ug BS=2 for Days 6 and 11, respectively. Furthermore, it is apparent that on Day 8 greater numbers of IgM PFC were inhibited by lower concentrations of BSA than were inhibited on Days 6 or 11 suggesting that the Day 8 population
FIG. 1. Inhibition on I>ays 6, 8, and
of IgM 11 after
anti-USA primary
plaque formation immunization.
by US:\.
F’FC
obtained
for
i~~hibitic~n
208
ROSZMAN
100
IO ,,g
I BSA
0.5
ADDED
‘FIG. 2. Inhibition of IgG anti-BSA plaque formation on Days 6, 8, and 11 after primary immunization.
by BSA. PFC obtained for inhibition
of IgM PFC contained proportionately greater numbers of high avidity IgM PFC. Similar inhibition studies were carried out with IgG PFC populations obtained on various days after immunization. The results of three representative experiments are summarized in Fig. 2. There was a definite decrease in the Iso values with time indicating a temporal increase in IgG avidity. There is, furthermore, a narrowing of the concentration of BSA required to effect IgG PFC inhibition on Days 8 and 11. In addition, 0.5 pg of BSA inhibited none of the IgG PFC observed on Day 6 but inhibited 12 and 17% of those IgG PFC which appeared on Day 8 and 11, respectively. This suggests that the heterogeneity of the IgG PFC population was decreasing with time due to ‘the emergence of higher avidity IgG PFC. In Table 2 are presented the mean I 60values from 3-6 separate experiments for each day of the response examined by means of the plaque inhibition assay. The Iso values for IgG antibody-producing cells decreasedwith time after immunization while the Iso values for IgM antibody producing cells remained constant after immunization with the exception of a sharp decreaseon Day 8. Higher concentrations
AVIDITY
OF PRIMARY AS A FUNCTION
IcM
TABLE 2 AND IGG ANTIBODY OF TIME
AFTER
Days after
SECRETED
BY
IMMUNIZATION
Avidity*
immunization Iii9 5
6 7 8 9 10 11
12.9 14.6 15.3 4.7 11.7 12.5 9.7
zt f f f f f f
W
1.2b 1.9 3.6 0.5 2.7 2.5 2.3
a rg BSA required to inhibit 50% of PFC (160). b Expressed as mean f standard error of the mean.
6.2 5.9 4.6 4.5 3.0 3.5
f f + f f f
0.9 0.5 0.9 0.8 0.5 0.1
PFC
AVIDITY
OF
PRIMARY
IGM
AND
IGG
209
ANTIHODY
of USA were required to achieve 50% inhibition of the IgM antibody-producing cells than IgG antibody-producing cells. Analysis of IgM and IgG PFC subpopulations. A more accurate reflection of the temporal change in the avidity of PFC populations can be obtained by enumerating the number of PFC which are inhibited by successive antigen concentration intervals (8). The mean of the results of 3-6 separate experiments for the IgG component of the response is illustrated in Fig. 3. From Day 6 ‘to 7 there was an increase in the number of PFC in each avidity subgroup with the largest increases occurring in higher avidity subgroups (O-2 pg BSA and 8-32 pg BSr\). On Da! 8, there was a decrease in the total number of IgG PFC with the greatest decrease occurring in the lower avidity IgG PFC subgroups. In fact, the number of IgG PFC in the high avidity subgroups remains very similar to that observed on Day 7 while low avidity IgG PFC (32-80 pg BSA) have completely disappeared. On the subsequent days of the response, there was a progressively larger loss of IgG J’FC in the next lowest avidity subgroup (S-32 ,u,g BSA) resulting in a predominance in high avidity PFC agreeing with the observed decrease in 150 \&es (Table 2). Analysis of the avidity subpopulations of IgM PFC was performed and the mean of the results of the 3-6 separate experiments are shown in Fig. 4. Kelatively flat distribution profiles were observed on all days of the response examined indicating a high degree of heterogeneity in terms of the avidity of the antibody produced by the IgM PFC. An increase in the number of low avidity (32-80 pg BS:Z) as well as high avidity IgM PFC (O-2 yg BSA) was observed between Days 5 and 6. The ratio of high to low avidity IgM PFC was less than one from Days 5-7, increased to about four on Day 8 and then decreased to approximately
DAY t-6
2-a AVIDITY
SUBGROUPS
6-32 (~9 BSAI
32-80
FIG. 3. Number of IgG PFC in various avidity ,subgroups during
primary
response.
210
ROSZMAN
I'
' 0-2
I 2-8 AVIDITY
FIG. 4. Number of
IgM
PFC
I 32-80
8-32 SlJBGROUPSCpg
in various avidity
BSA)
subgroups
during
primary
response.
1.5 on Days 9-l 1. The avidity of the IgM synthesized by the PFC, however, remained heterogeneous throughout the measurement period and there was not a total disappearance of low avidity IgM PFC as noted in the case of the IgG response. DISCUSSION In the present study the avidity of IgM and IgG antibody produced by PFC during the primary antibody response to BSA was analyzed employing a plaque inhibition assay. The results demonstrated that Ithe avidity of IgG antibody increased during the response and that the increase was due to attrition of low avidity producing PFC. The net result of this is the eventual dominance of cells secreting higher avidity IgG antibody. Thus, these results demonstrate that maturation of the IgG antibody response does occur and are in agreement with observations of others (2-6). There are reports indicating that the antigen-binding characteristics of IgM antibody does increase after immunization (9, 10, 13, 15), whereas others have reported that it does not increase (4, 11, 12, 14). Thus, it is not firmly established whether or not the IgM antibody response is operating under an antigen-driven maturation process similar to that observed for IgG antibody. While these studies were in progress, Claflin et al. (16) measured the temporal change in the avidity of primary IgM and IgG anti-hapten antibody employing a plaque inhibition assay and observed that the avidity of IgM increased with time. The results of my study are more consistent with the notion that the avidity of IgM antibody does not increase markedly during the primary antibody response to BSA. There was a striking increase on Day 8 in the avidity of IgM antibody as evidenced by the decrease in the Lo value (Table 2). This increase in avidity clearly results from the fact that there
are approximately fourfold more high avidity IgM I’FC present than low avidit> IgA4 PFC (Fig. 4) either as a result of the emergence of high avidity IgAl 1’lY or the more rapid disappearance of low avidity IgM PFC. This obser\-ation ~voul(l seem to argue that the avidity of IgM antibody does increase during the primar-! response but for the following reasons this argument does not appear to be ten:lble. First, during the period when the number of TgM PFC was increasing, thcrct was a greater increase in the number of low avidity TgM PFC as colnparetl to high avidity IgM PFC. Second, on Days 9-11 the ratio of high avidity to low avidity IgM PFC was not maintained at the level observed on Day 8 bllt in fact decreased. Third, the low avidity IgM PFC do not disappear from the resl~onsc with time as was the case for the IgG response. Finally, analysis of the TghT I’lX: avidity subpopulations revealed substantial heterogeneity throughout the mcasur~ment period in contrast to the findings of Claflin ct al. (16) which denlonstrntetl a time-related shift from a heterogeneous population of IgM PFC to a more hntnogeneous population consisting of predominantly higher avidity IgM PFC. 7‘hese observations taken together, therefore, indicate that the avidity of IgM nntil)otl!, relllains constant throughout the primary antibodv response to TSS:1 and it is cow chided that antigeti-driven maturation is not a major feature of the IgRI responw with the concentration of antigen employed in this study. Although the results of this study and those of Claflin c’t 01. (16) arc newt in agreement this may in part be due to the difference in animal species (ral)lGt vs mouse) and the antigens employed (hapten vs HS.1). It is also conceivable that antigen concentrations different from those employed in the present stutly could lend to maturation. Wu and Cinader (1 5) have reported, holvever. an increase ill the aftinity of rabbit primary IgM antibody after immunization with a corlcc~~trntion of antigen identical to that employed in this stu(l!-.
This author
investigation thanks Mike
\\as supported in part by a grant from hfyrick for his expert technical as+istancc.
tlw
IZescarcl~
Clorpor;itioll.
.1‘11(
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 0. 10. Il. 12. 1.i. 1-l. 15. 16. 17. 1X. 19.
Siskind, G. W., and Benacerraf, B., Adrw. Imwrtr~ol. 10, 1, 196X. Eisen. H., and Siskind, G. W., Bioclwmisfv~r 3, 996. 1904. Siskind, G. W., Dunn, P., and Walker, J. G., J. E.rp. Jlrtf. 127. 55, 1968. Sarvas, IT., and Makela, O., Illl11z111zOchrllliSfYY 7, 933. 1070. Miller, G. W., and Segre, D., J. Zwm~ol. 109. 74. 1072. .\ndersson. B., J. Escp. Med. 135, 312, 1972. Antlersson, B., J. Exj. Med. 131, 77, 1970. Davie. J. M., and Paul, W. E., 1. Exp. Xcd. 135, 660. 1072. Finkelstein, hi. S., and Uhr, J. W., J. Imwurxol. 97, 56.5, 1906. M’ehster, K. G., Iwmunologjt 14, 39, 1968. Baker, P. J.. Prescott, B., Stashak, 1’. \V., and Am&augll. I). I;.. .I. fwu~ux~rl. 1071. C‘lcm, Is. \I‘., and Small, P. A., J. Exp. Arcd. 132, 385, 1070. Claflin. I,., Merchant, B., Cell. 1w~zu)zo/. 5, 209. 1972. Gunn. D. T... and Roszman, T. L., J. Iwmzrruol. Jirth~uls 1, 381, 1072. \I,‘u, C. Y., and Cinader, B., Ew. J. Im~~wo1. 2, 39X. 1972. Clafin. L.. Merchant, B., and Inman, J., J. Iu~wu~lol. 110, 241. 1973. Roszman, 7‘. I*., Folds, J. D., and Stavitsky, A. B.. 1uzw1~010~~~, 20, 1041, 1971. Cunningham, A. J., and Szenberg, A., Imrmrnolo~g~ 14, 599, 1968. Nordin, A. A., Cosenza, A., and Hopkins, W., J. Imnwwl. 103, 859, 1969.
107.
710,
IMMUNOLOGY
Analysis
205-211
11,
(1974)
of the Temporal Changes in the Avidity of Primary IgM and IgG Antibody at the Cellular Level T. L. ROSZMAN
Department
of
Cell
Biology, College of Medicine, Lexington, Kentucky 4506 Received
Jwne
University
of Kentucky,
29, 1973
The avidity of primary IgM and IgG anti-bovine serum albumin antibody was measured at the cellular level. The avidity of IgG antibody secreted by plaqueforming cells increased with time as evidenced by the fact that progressively less bovine serum albumin was required to inhibit 50’76 of the plaque-forming cells. Analysis of various avidity subpopulations of IgG plaque-forming cells, furthermore, demonstrated that there was a temporal attrition of low avidity IgG plaque-forming cells resulting in the dominance of high avidity IgG plaque-forming cells. Thus,
maturation of IgG antibody was occurring during the primary antibody response to bovine serum albumin. Similar analyses of the IgM antibody secreted by plaqueforming cells indicated that the avidity the exception of a marked increase
subpopulations ing
that
remained
constant
throughout
the response
with
on Day 8. Heterogeneity of the various avidity of IgRI plaque-forming cells was a hallmark of the response indicat-
maturation
is not
a major
feature
of the IghI
response.
IXTRODUCTION It is well known that a temporal increase in the antigen binding characteristics of IgG antibody occurs after primary immunization as a result of antigen preferentially selecting those precursors of the antibody-forming cells with higher affinity receptors (1). Maturation of the IgG response has been observed at the humoral (24) as well as the cellular level (5, 6). Andersson (7) has demonstrated, employing the plaque inhibition assay, that the time-related increases in the avidity of IgG antibody secreted by plaque-forming cells (PFC) is similar to that observed in the serum. A correlation, furthermore, was found to exist between the rate of increase in the avidity of IgG antibody at the serum and cellular level with the increase
avidity
of antigen-binding
cells during
the immune
response
to dinitro-
phenol-guinea pig albumin (8). Similar studies concerned with determining whether or not the IgM responses undergoes antigen-driven maturation have yielded conflicting results (9-15). Recently, however, Claflin et al. (16j have observed that the avidity of primary IgM antibody produced by PFC does increase with time and that the maturation process is complete by 9 to 10 days after primary immunization. In the present study, the temporal changes in the avidity of IgM and IgG antibody secreted by PFC during the primary antibody response to bovine serum albumin (BSA) was examined employing a plaque inhibition assay. The results 205 Copyri ht Q 1974 by Academic Press, All rig e;ts of reproduction in any form
Inc. reserved.
206
ROSZMAN
are consistent with the conclusion that a maturation process is operative as evidenced by the fact that the avidity of IgG antibody does increase with time. The kinetics of development of the avidity of IgM antibody differed considerably from ‘that of IgG antibody which indicates that antigen-driven maturation is not a feature of the IgM primary antibody response. MATERIALS
AND
METHODS
Animals and immunization. Female New Zealand white rabbits 3-6 months of age were immunized with 0.5 mg of BSA (Miles Laboratories, Inc., Kankakee, IL) incorporated into complete Freund’s adjuvant (Difco Labs, Detroit, MI) in each hind footpad. Rabbits were sacrificed at various times after primary immunization and popliteal lymph nodes removed. Preparation of cell suspensions. Cell suspensions were prepared from the spleens and popliteal lymph nodes according to the method of Roszman et al. (17). The final concentration of the nucleated cells was adjusted to l-2 x log/ml in Eagle’s minimum essential medium (MEM, Grand Island Biological Co., Grand Island, NY). Preparation of sensitized sheep red blood cells. The carbodiimide method of Gunn and Roszman (14) was employed to couple BSA to sheep red blood cells (SRBC). Cells were coupled in the presence of 100 mg/ml of BSA since preliminary experiments demonstrated that maximal numbers of both IgM and IgG PFC were detected when this concentration of protein was used for coupling. The sensitized SRBC were resuspended to a final concentration of 7% (v/v) in MEM. Plaque assay technique. A modification of the Cunningham and Szenberg liquid plaque assay (18) was employed. Two siliconized microscope slides were taped together with double side tape (Dubl-Stik Tape, Kleen-Stik Prods., Newark, NJ) to form two chambers with a volume of approximately 0.05 ml each. Immunoglobulin G AFC were developed by facilitation with a goat anti-rabbit IgG serum (Cappel Laboratory, Downingtown, PA) at a final dilution of 1 : 50. This facilitating antiserum caused complete inhibition of IgM AFC. The plaquing chambers were incubated at 37°C for 30-60 min to develop IgM AFC and 90-120 min for IgG AFC. After incubation, the plaques were counted under 35 X magnification. In addition to developing more rapidly, IgM PFC were completely inhibited by concanavalin A (19)) whereas IgG PFC were not. Plaque inhibition assay technique. The method of Andersson (7) was employed to determine the percent inhibition of IgM and IgG PFC by various concentrations of free BSA. A mixture containing guinea pig complement, BSA sensitized SRBC, facilitating antiserum when needed, and an aliquot of lymphoid cells containing between 400 and 800 PFC/O.l ml was prepared. A 0.1 ml aliquot of this was added to each of a series of tubes containing 0.1 ml of increasing concentraItions of BSA in MEM ranging from 0.1 to 100 pg. Duplicate chambers were filled with these mixtures and the number of PFC determined as previously described. That concentration of BSA which inhibited 50% of PFC was taken as the avidity (ho). Analysis of avidity subpopula’tions. The method of Davies and Paul (8) was employed to determine the absolute number of IgM and IgG PFC in each avidity subgroup.
AVIDITY
01’
Days after immunization
PRIMARY
IGM
AND
Number
IGG
of I’FC,‘lV
AS7‘1
HOI)\
nucleated
cells
1gn1
.i
212 f 108.1 f
6
i x 0 10 11
132” 115
41 It22
935
zk 190
974 1725
185
r!z 36
1166
113 39
f
f- 141 108.3 * 506 2% f 102
16
l
16
.X3 rt
16
f f
257 .362
i
230
676
RESULTS Kinetics of the IgM and IgG pviuzary antibody respomc. The appearance of IgM and IgG PFC in the popliteal lymph nodes at various times after a single injection of antigen is illustrated in Table 1. The results represent the mean of j-7 separate experiments. There is a rapid rise and decline of IgM and IgG PFC with maximum numbers of IgG PFC appearing one day after the IgM peak. Average avidity of IgM and IgG atztibody secreted by PFC. Inhibition experiments were performed in order to determine the temporal change in the avidity of IgM and IgG antibody synthesized during the primary antibody response. The data from three representative experiments showing inhibition of the IgM I’FC are presented in Fig. 1. The highest avidity IgM antibody was produced on Day S of the response with an 150value of 4.5 pg BSA as compared to Ir,o values of 15.1 and 11.5 ,ug BS=2 for Days 6 and 11, respectively. Furthermore, it is apparent that on Day 8 greater numbers of IgM PFC were inhibited by lower concentrations of BSA than were inhibited on Days 6 or 11 suggesting that the Day 8 population
FIG. 1. Inhibition on I>ays 6, 8, and
of IgM 11 after
anti-USA primary
plaque formation immunization.
by US:\.
F’FC
obtained
for
i~~hibitic~n
208
ROSZMAN
100
IO ,,g
I BSA
0.5
ADDED
‘FIG. 2. Inhibition of IgG anti-BSA plaque formation on Days 6, 8, and 11 after primary immunization.
by BSA. PFC obtained for inhibition
of IgM PFC contained proportionately greater numbers of high avidity IgM PFC. Similar inhibition studies were carried out with IgG PFC populations obtained on various days after immunization. The results of three representative experiments are summarized in Fig. 2. There was a definite decrease in the Iso values with time indicating a temporal increase in IgG avidity. There is, furthermore, a narrowing of the concentration of BSA required to effect IgG PFC inhibition on Days 8 and 11. In addition, 0.5 pg of BSA inhibited none of the IgG PFC observed on Day 6 but inhibited 12 and 17% of those IgG PFC which appeared on Day 8 and 11, respectively. This suggests that the heterogeneity of the IgG PFC population was decreasing with time due to ‘the emergence of higher avidity IgG PFC. In Table 2 are presented the mean I 60values from 3-6 separate experiments for each day of the response examined by means of the plaque inhibition assay. The Iso values for IgG antibody-producing cells decreasedwith time after immunization while the Iso values for IgM antibody producing cells remained constant after immunization with the exception of a sharp decreaseon Day 8. Higher concentrations
AVIDITY
OF PRIMARY AS A FUNCTION
IcM
TABLE 2 AND IGG ANTIBODY OF TIME
AFTER
Days after
SECRETED
BY
IMMUNIZATION
Avidity*
immunization Iii9 5
6 7 8 9 10 11
12.9 14.6 15.3 4.7 11.7 12.5 9.7
zt f f f f f f
W
1.2b 1.9 3.6 0.5 2.7 2.5 2.3
a rg BSA required to inhibit 50% of PFC (160). b Expressed as mean f standard error of the mean.
6.2 5.9 4.6 4.5 3.0 3.5
f f + f f f
0.9 0.5 0.9 0.8 0.5 0.1
PFC
AVIDITY
OF
PRIMARY
IGM
AND
IGG
209
ANTIHODY
of USA were required to achieve 50% inhibition of the IgM antibody-producing cells than IgG antibody-producing cells. Analysis of IgM and IgG PFC subpopulations. A more accurate reflection of the temporal change in the avidity of PFC populations can be obtained by enumerating the number of PFC which are inhibited by successive antigen concentration intervals (8). The mean of the results of 3-6 separate experiments for the IgG component of the response is illustrated in Fig. 3. From Day 6 ‘to 7 there was an increase in the number of PFC in each avidity subgroup with the largest increases occurring in higher avidity subgroups (O-2 pg BSA and 8-32 pg BSr\). On Da! 8, there was a decrease in the total number of IgG PFC with the greatest decrease occurring in the lower avidity IgG PFC subgroups. In fact, the number of IgG PFC in the high avidity subgroups remains very similar to that observed on Day 7 while low avidity IgG PFC (32-80 pg BSA) have completely disappeared. On the subsequent days of the response, there was a progressively larger loss of IgG J’FC in the next lowest avidity subgroup (S-32 ,u,g BSA) resulting in a predominance in high avidity PFC agreeing with the observed decrease in 150 \&es (Table 2). Analysis of the avidity subpopulations of IgM PFC was performed and the mean of the results of the 3-6 separate experiments are shown in Fig. 4. Kelatively flat distribution profiles were observed on all days of the response examined indicating a high degree of heterogeneity in terms of the avidity of the antibody produced by the IgM PFC. An increase in the number of low avidity (32-80 pg BS:Z) as well as high avidity IgM PFC (O-2 yg BSA) was observed between Days 5 and 6. The ratio of high to low avidity IgM PFC was less than one from Days 5-7, increased to about four on Day 8 and then decreased to approximately
DAY t-6
2-a AVIDITY
SUBGROUPS
6-32 (~9 BSAI
32-80
FIG. 3. Number of IgG PFC in various avidity ,subgroups during
primary
response.
210
ROSZMAN
I'
' 0-2
I 2-8 AVIDITY
FIG. 4. Number of
IgM
PFC
I 32-80
8-32 SlJBGROUPSCpg
in various avidity
BSA)
subgroups
during
primary
response.
1.5 on Days 9-l 1. The avidity of the IgM synthesized by the PFC, however, remained heterogeneous throughout the measurement period and there was not a total disappearance of low avidity IgM PFC as noted in the case of the IgG response. DISCUSSION In the present study the avidity of IgM and IgG antibody produced by PFC during the primary antibody response to BSA was analyzed employing a plaque inhibition assay. The results demonstrated that Ithe avidity of IgG antibody increased during the response and that the increase was due to attrition of low avidity producing PFC. The net result of this is the eventual dominance of cells secreting higher avidity IgG antibody. Thus, these results demonstrate that maturation of the IgG antibody response does occur and are in agreement with observations of others (2-6). There are reports indicating that the antigen-binding characteristics of IgM antibody does increase after immunization (9, 10, 13, 15), whereas others have reported that it does not increase (4, 11, 12, 14). Thus, it is not firmly established whether or not the IgM antibody response is operating under an antigen-driven maturation process similar to that observed for IgG antibody. While these studies were in progress, Claflin et al. (16) measured the temporal change in the avidity of primary IgM and IgG anti-hapten antibody employing a plaque inhibition assay and observed that the avidity of IgM increased with time. The results of my study are more consistent with the notion that the avidity of IgM antibody does not increase markedly during the primary antibody response to BSA. There was a striking increase on Day 8 in the avidity of IgM antibody as evidenced by the decrease in the Lo value (Table 2). This increase in avidity clearly results from the fact that there
are approximately fourfold more high avidity IgM I’FC present than low avidit> IgA4 PFC (Fig. 4) either as a result of the emergence of high avidity IgAl 1’lY or the more rapid disappearance of low avidity IgM PFC. This obser\-ation ~voul(l seem to argue that the avidity of IgM antibody does increase during the primar-! response but for the following reasons this argument does not appear to be ten:lble. First, during the period when the number of TgM PFC was increasing, thcrct was a greater increase in the number of low avidity TgM PFC as colnparetl to high avidity IgM PFC. Second, on Days 9-11 the ratio of high avidity to low avidity IgM PFC was not maintained at the level observed on Day 8 bllt in fact decreased. Third, the low avidity IgM PFC do not disappear from the resl~onsc with time as was the case for the IgG response. Finally, analysis of the TghT I’lX: avidity subpopulations revealed substantial heterogeneity throughout the mcasur~ment period in contrast to the findings of Claflin ct al. (16) which denlonstrntetl a time-related shift from a heterogeneous population of IgM PFC to a more hntnogeneous population consisting of predominantly higher avidity IgM PFC. 7‘hese observations taken together, therefore, indicate that the avidity of IgM nntil)otl!, relllains constant throughout the primary antibodv response to TSS:1 and it is cow chided that antigeti-driven maturation is not a major feature of the IgRI responw with the concentration of antigen employed in this study. Although the results of this study and those of Claflin c’t 01. (16) arc newt in agreement this may in part be due to the difference in animal species (ral)lGt vs mouse) and the antigens employed (hapten vs HS.1). It is also conceivable that antigen concentrations different from those employed in the present stutly could lend to maturation. Wu and Cinader (1 5) have reported, holvever. an increase ill the aftinity of rabbit primary IgM antibody after immunization with a corlcc~~trntion of antigen identical to that employed in this stu(l!-.
This author
investigation thanks Mike
\\as supported in part by a grant from hfyrick for his expert technical as+istancc.
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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 0. 10. Il. 12. 1.i. 1-l. 15. 16. 17. 1X. 19.
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