Volume 7, number 3

MOLECULAR• CELLULARBIOCHEMISTRY

June 30, 1975

THE GENETIC CONTROL OF THE ANTIBODY RESPONSE IN INBRED RATS* Sandra K. RUSCETTI, Thomas J. GILL III and Heinz W. KUNZ

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 (Received August 8, 1974)

Summary

Introduction

The antibody response of genetically inbred rats to poly(Glu52Lys33Tyr15) is controlled by a compleX polygenic system which includes at least two autosomal genes and a sex influence, which may also be genetically determined. The genetic control of the quantity, binding constants, and specificity of the antibody formed is linked to the major histocompatibility locus. Factors other than the major genetic ones and the sex influence also affect the quantity of antibody formed, since animals of the same genotype can make significantly different amounts of antibody, depending upon the crosses by which they acquire the major histocompatibility alleles. After immunization with poly(Glu52Lys33Tyr15) the low responders make fewer antibody-producing cells, are not capable of mounting a delayed hypersensitivity reaction to the polypeptide and appear to be deficient in their ability to produce the specific IgM antibody. Immunization of the low responders with antigen aggregated with methylated bovine serum albumin enhances the quantity of antibody formed, increases the binding constants and crossreactivity of the antibody and enhances the delayed hypersensitivity response. In contrast to the findings with the L-amino acid polypeptide, there does not appear to be any genetic control over the antibody response to the D-amino acid enantiomorph poly(oGlu52oLys33oTyrlS), which is minimal in all strains.

The ability of an animal to mount an immune response depends upon the interplay between the chemistry of the antigen and the physiological state of the host and upon the presence of structural and regulatory genes necessary to produce immunoglobulin molecules specific for the antigen (Fig. 1). Although structural genes, which determine the particular sequence of amino acids present in an immunoglobulin molecule, are of obvious importance, the role of regulatory genes in the development of the immune response has been increasingly realized. Many animals possessing the necessary structural genes to code for a specific immunoglobulin molecule produce a barely detectable humoral antibody response to the appropriate antigen. These animals are thought to lack the proper immune response genes which regulate whether antibodies will be produced to a particular antigen. Two types of small lymphocytes are involved in the development of an antibody response: the

* An invited article.

.........

y:

Ab Ag complex

ANTIGEN ANTIBODY

/

SHEM BTRY structure omount time c~fobcllsm

MEMBRANE STRUCTURE receptor sites(R} metabohsm

\

GENETICS number of cells progrommlng

]

SPECIFICITY onfibody 51~e5

Fig. 1. Schematic representation of the hypothesis about the induction and genetic control of the i m m u n e response.

Dr. W. Junk b.v: Publishers - The Hague, The Netherlands

145

TABLE I Genetically controlled immune responses to specific antigens and mitogens ALLOTYPE LINKED REFERENCES A. Mouse c~1-3 dextran 6 7 Poly(Ser), Poly(Ala) 8,9 Phosphorylcholine 10 Tobacco mosaic virus Serum agglutinin production to SRBC 11,12 Para-aminobenzoic acid-bovine 13, 14 v-globulin Streptococcal A and C carbohydrate 15,16 B. Humans Rh blood groups Salmonella flagellin II. HISTOCOMPATIBILITY LINKED A. Mouse Poly(Tyr, Glu)-poly(DLAla)--poly(Lys) Poly(Glu 6oAla3oTyr i 0), poly(Glu57Lys38Ala 5) Poly(Glu58Lys38Phe 4) Bovine serum albumin, ovalbumin, ovomucoid Trinitrophenyl-mouse serum albumin Male histocompatibility antigen (H-Y) IgA and IgG myeloma H-2.2 antigen H-13 antigen Theta AKR (Thy. 1.1) antigen Thyroglobulin Gross, Friend viruses Lymphocytic choriomeningitis virus Mammary tumor virus Mouse Ea-1 blood groups Lactic dehydrogenase Staphylococcal nuclease B. Guinea Pig Polylysine, DNP-polylysine, polyalanine Poly(Glu6°Ala4°), poly(GluS°Tyrs°) Bovine serum albumin, human serum albumin Poly(Tyr-Glu-Ala-Gly) Dinitrophenyl-guinea pig albumin Glucagon-polylysine

146

17 18

19, 20 21,22 23 24, 25 26 27 29 30,31 32, 33 34, 35 36,37 38 39,40 41 42 43,44 45 46 19,20 47,48

E. Rabbit Poly(Glu 56Lys 38Tyr6)

65,66

F. Chicken Tuberculin sensitivity

67

III. X-CHROMOSOME LINKED A. Mouse • Type III pneumococcal polysaccharide Synthetic double-stranded RNA

68, 69 70

IV. NOT HISTOCOMPATIBILITY LINKED A. Mouse Poly(Tyr, Glu)-poly(Pro)--poly(Lys) Reagin production Cross-reactivity to lipopolysaccharide Streptococcal A carbohydrate Keyhole limpet hemoeyanin Loop region of lysozyme

4, 71, 72 73, 74 75 76, 77 78 79, 80

B. Guinea Pig Hydralazine C. Rat Coneanavalin A and phytohemagglutinin stimulation Poly(Glu6°Ala3°Tyrx°) V.

LINKAGE UNKNOWN A. Mouse Fd bacteriophage Sheep red blood cells Tolerance to bovine serum albumin ,/G2, allotype Allogeneic bone marrow Coneanavalin A and phytohemagglutin stimulation Denatured DNA/methylated bovine serum albumin

81

82, 83 84

85, 86 87, 88 89 90 91 92, 93

49,50 51 50, 52 53

B. Guinea Pig Insulin

C. Rat Lactic dehydrogenase Poly(Tyr,Glu)-poly(DLAla)--poly(Lys) Poly(GluS2Lys33Tyr a5) Encephalitogenic protein

54,55 56, 57 58,59 60,61

C. Rabbit Poly(Tyr,Glu)-poly(DLAla) poly(Lys) Bovine serum albumin Lactic dehydrogenase Group A, C streptoeoceal carbohydrates

97 89, 98 99 100

D. Humans Ragweed antigen E Ragweed allergen Ra5 Bacterial antigens

62 63 64

D. Chicken Graft-versus-host reaction Blood group A Dinitrophenyl-chicken gamma globulin

101 102 103,104

94 95, 96

B-lymphocyte* which is thought to contain the immunoglobulin structural genes, and the Tlymphocyte, which is thought to be necessary as a helper or regulator cell in the development of an immune response to m a n y antigens. There have been a variety of proposals dealing with the possible ways in which the two types of lymphocytes, and third cell type, the macrophage, could cooperate. The latter might cooperate with the T-lymphocyte by concentrating antigen or antibody-antigen complexes shed by the T.-cells on its surface and subsequently providing lbr the proper stimulation of B-lymphocytes for the production of specific antibody molecules ~. I m m u n e response genes could be expressed at the level of any of the three cell types, but current thinking favors the T-cell as the major site of expression 2,3. M a n y studies have been carried out within the last 10 years to determine why certain animals produce high concentrations of antibody to a particular antigen whereas others do not. The antibody responses to a variety of antigens are governed by immune response genes 2'4's, and they are summarized in Table t. Differences in antigen metabolism do not account for the genetically controlled differences in the antibody response ~05, although the persistence of an antigen in the tissues influences its ability either to stimulate an immune response or to induce tolerance 1°6. Both high and low responding strains usually possess the necessary structural genes for a given antibody, although the low responders may have fewer B-lymphocytes expressing the structural genes 4. Various studies on the function of the immune response genes suggest that: (a) they may code for antigen receptors on the T-cell and may play a role in antigen recognition 2,3 ,107; (b) they may code for a * The abbreviations used in this paper are: DNP, dinitrophenyl; SRBC, sheep red blood cells; T-cell, thymus-derived lymphocyte; B-cell, bone marrow-derivedlymphocyte; PFC, plaque-formingcell; MeBSA, methylated bovine serum albumin; Kb, average binding constant; Ab, antibody; Ag, antigen; Ag-B, major histocompatibilitylocus of the rat; H-2, major histocompatibilitylocus of the mouse; HL-A, major histocompatibility locus of humans. The nomenclature of the synthetic polypeptides is defined according to the rules on Abbreviated Nomenclature of Synthetic Polypeptides (J. Biol. Chem. 243, 2451,1968). The superscript following each amino acid denotes the mole percentage of that amino acid in the random, linear copolymer. All amino acid residues in the peptides studied are in the L-configuration, except where the contrary is stated.

protein involved in regulating the interaction of T-cells with either B-cells 1°8 or with macrophages lO9,110; or (c) they can be expressed solely at the B-cell 4'6'13'111 or macrophage 112 level. A number of investigations have been undertaken to elucidate the role of these immune response genes, and each study has contributed valuable information to our understanding of the regulation of the immune response. In our laboratory, we have utilized a synthetic polypeptide antigen to study the various parameters controlling the antibody response in the inbred rat, and this paper will review these studies.

Genetic control of the antibody response to poly(Glu52Lys33Tyr 15) in inbred rats Br eedin9 ,studies The antibody response to the linear synthetic polypeptide poly(Glu52Lys33Tyr 1s) (molecular weight = 28,000 daltons) was studied in various strains of rats and in their hybrids by assaying for antibody with a quantitative immunoadsorbent micromethod a 13, which can detect as little as 10 #g of antibody per ml of serum. The secondary response after immunizing with antigen in complete Freund's adjuvant and then in aqueous solution was studied. Certain strains of rats, such as the ACI strain, were high responders, producing greater than 500-800 #g of antibody per ml of serum, whereas others, including the F344 strain, were low responders, producing less than 200 #g of antibody per ml. Breeding studies showed that the immune response to this antigen was under the control of at least two autosomal genes 59,114. The antibody response of the F 1 hybrid between the high and low responders was intermediate between the responses of the parental strains but skewed toward low responses. The F 2 generation showed a broader distribution in the magnitude of the antibody responses and segretion into populations of low, moderate and high responders. The backcrosses of the F 1 hybrid to the high responder parental strain showed mainly moderate and/or high responders and only a few low responders. The backcross to the low responding parental strain showed a large number of low responders and some moderate and high responders. The backcrosses having a low antibody response bred true with inbreeding and with second backcrossing, so they apparently had only those genetic factors that lead to a low antibody 147

response. Inbreeding studies with the highly responding backcross animals showed that they also bred true. The moderately responding backcrosses gave offspring that showed the whole spectrum of antibody responses, as would be expected for control by multiple genetic factors. By assigning the animals to low, moderate and high responder categories, the ratios among the various categories determined experimentally. were compared to the theoretical predictions for various gene models (Fig. 2). Other approaches were also taken to analyze the experimental results, and a model involving only one gene would not fit the data. Therefore, it was concluded that the immune response to poly(GluSZLysaaTyr aS) was under the control of at least two autsomal genes. At least one of the genes governing the immune response to poly(GluSZLysa3Tyr is) is linked to the major histocompatibility locus of the rat (Ag-B or H-l) a~5. Low responsiveness is associated with Ag-B types 1, 3 and 6; high responsiveness, with types 4 and 5; and moderate responsiveness with type 2 (Fig. 3). Breeding studies, such as the one in Figure 4, confirm an association of Ag-B type with the level of antibody produced, as do the following subsidiary lines of evidence: (1) all representatives of each Ag-B group behaved the same with respect to antibody formation; (2) genetically uncharacterized low THEORETICAL

EXPERIMENTAL

,Ag-B I

Ag-B2

Ag-B3

Ag-B4

Ag-B5

Ag-B6

1500 1250 iooo I"I

750

I

500

'!

Jil

Fig. 3. Bar histogram showing the gntibody response to poly(Glu52Lys33Tyr ~5) in inbred rats belonging to the various Ag-B groups.

responder strains behaved the same in the linkage studies as the genetically defined low responding strains; and (3) the broad antibody response in a population of one strain of rats (NBR) was due to the presence of both low and moderate responders (Ag-B1 and Ag-B2). This linkage was confirmed by GONTHERet al) ~6, who showed that a congenic rat strain in which the Ag-B4 (high responder) allele was put on an Ag-B1 (low responder) background was a high responder to

I

I.O 0.8

~o8

~06

,Ill II,I ],1I,il

0 LMH MH LMH LMH LMH LMH AC! F344 FA FA/FA FA/A FA/F [L-800,ug Ab/m,

j

Fig. 2. Frequency histogram comparing the experimental results and theoretical calculations for the hypothesis that two independently segregating genes control the antibody response to poly(Glu52LysaaTyr~S). The ACI strain is a high responder and the F344 strain is a low responder. The categories of response are: L (low), ~800 fig Ab/ml. The details of the genetic calculations are given in reference 59. In designating the various strain combinations, A is used for the ACI strain and F for the F344 strain. The first symbol in a hybrid denotes the maternal strain. The symbol preceding the slash in the backcrosses also denotes the maternal strain.

148

%0.6

0 LMH ACT

LMH S5B

Ag-B 4,4 r.,'/x,'J Ag - B I, 4 I Ag-B I,I

LMH AS

LMH AS/AS

LMH AS/A

L MH AS/S

CATEGORIES OF RESPONSE FOR EACH GROUP

Fig. 4. Bar histogram comparing Ag-B type with the frequency of antibody response in high, moderate and low categories. L = ~ 800/~g Ab/ml. In designating the various strain combinations, A is used for the ACI strain and S, for the S5B strain.

poly(Glu52Lys33TyrlS). Genetic factors other than those controlling the ability to respond influence the quantity of antibody formed, because the amounts of antibody made by rats having the same Ag-B genotype but having acquired the major histocompatibility alleles by different breeding schemes are often significantly different. These factors may be associated with minor histocompatibility or mixed lymphocyte reactivity loci1 t 5. The antibody response to poly(Glu52Lys33Tyr 15) is sex-influenced 59'1 ~4,a15. There are generally more high responders among females and more lower responders among males (Fig. 3), and the magnitudes of the antibody responses are more heterogeneous in the females except for those of the poorly responding strains. In order to determine whether sex hormones were playing a significant role in this sex influence, the effects of gonadectomy were studied 1aT. Gonadectomy did not alter the antibody response to poly(Glu52Lys33Tyrl 5) in either males or females, regardless of the age at which it was performed (10 days or 9 weeks) or the interval between gonadectomy and immunization (1 week or 10 weeks). These findings suggest that the higher female antibody response in our experimental system was not due to the influence of sex hormones. The mechanism which mediates the sex differences in the antibody response is still not clearly defined, but it may involve another gene which is linked to the X-chromosome.

Antibody response to the D-isomer There is no evidence for genetic control of the antibody response to the D-amino acid polypeptide poly(DGluS2DLys33DTyr15)118, in contrast to the findings with the L-isomer. All rats tested were low responders to this antigen (Fig. 5). There is no cross-reactivity between the isomers at the level of the induction of the immune response Its, even though there is some immunochemical cross-reactivity119. Effect of aggregation on antibody formation When poly(GluS2LysS3Tyr 15) is complexed with a macromolecule of opposite charge, such as methylated bovine serum albumin, and rats are immunized with this aggregate, the antibody response to poly (Glu52Lys33Tyr is) in the poorly responding strains is enhanced 2-7 fold, while the antibody response to poly(Glu52Lys33Tyr15)

in the highly responding strains decreases 2-4 fold59,118,12o (Fig. 5). The antibody response to the D-isomer is enhanced 4-10 fold in all strains, except the moderately responding WF strain. There are two possible mechanisms by which aggregation may affect the antibody response, and these mechanisms need not be mutually exclusive. The aggregate may affect the interaction of the antigen with the appropriate T-cells. In those cases in which it increased the antibody response, it may be facilitating the reaction of the antigen with low affinity receptors on the T-cell surface. When the aggregate decreased the antibody response in the high responding strain, it may have reacted with T-cells which have high affinity receptors for the antigen in such a fashion as to prevent their subsequent interaction with B-cells. Alternatively, the aggregate may function by regulating the release of antigen and by protecting the antigen from enzymatic degradation. If the antibody response is normally low, the slow release of small amounts of antigen may stimulate the maximal number of cells capable of reacting with it and minimize the induction of tolerance. The prolonged release of antigen could cause partial tolerance in the highly responding strains, whose Ag-BI

Ag-B2

Ag-B3

Ag-B4

Ag-B5

Ag-B6

tSO0 1250

14 I000

750

- 500

~ ~~ i

%I

~,

250

D.n 1, LEW

WE

.n ,n 0.n

BN ACT RAT STRAINS

AUG

BUF

Fig. 5. Bar histogram showing the antibody response of inbred rats belonging to the various Ag-B groups to: poly(Glu 52Lys33Tyrl 5) (L-isomer), ~ ; poly(Glu52Lys33Tyr 15)/MeBSA (aggregated L-isomer), ~ ; poly(DGluS2DLys33DTyr 15) (D-isomer), ~ ; and poly(DGlu52oLys33DTyrlS)/MeBSA (aggregated D-isomer), ~ . MeBSA is methylated bovine serum albumin.

149

Table 2 Delayed hypersensitivity reaction to poly(GluS2Lys33Tyr 15) alone or aggregated with methylated bovine serum albumin I 2o Skin test antigen* Strain ACI (high responder)

F344 (low responder)

Immunizing antigen

Poly(Glu52Lys33Tyr 15)

Poly(Glu52Lys33Tyr 15)/MeBSA

poly(Glu 52Lys33Tyrl 5) poly(Glu 52Lys33Tyrl 5)/MeBSA

++ ++

++ ++

poly(Glu52Lys33Tyr 15) poly(Glu52Lys33Tyrl 5)/MeBSA

0

_+

0

++

* 100 #g of antigen was used for the skin test, and the tests are scored on a scale of 0 to + +

cells react well with the antigen, and lead to a decrease in the amount of antibody formed.

Delayed hypersensitivity response The ability to mount a delayed response against poly(Glu52Lys33Tyr 15) can be correlated with the ability to produce antibodies against the polypeptide (Table 2). After immunization with either aggregated or unaggregated antigen, the highly responding strain showed a strong delayed hypersensitivity reaction when skin tested with the immunizing antigen. In contrast, only the low responding rats that had been immunized with antigen aggregated with methylated bovine serum albumin showed a delayed reaction.

Antibody-forming cell response The kinetics of antibody formation at the cellular level were studied in both high and low responding strains. Both strains produced only a few antibody-forming cells (20 PFC/106 spleen cells) after immunization with poly(Glu 52Lys33Tyr15) in complete Freund's adjuvant 12~'122. After secondary stimulation with antigen, the highly responding ACI strain produced a large number of IgM producing cells in the spleen (500-1200/ 10 6 spleen cells), whereas there was no increase in the low responding F344 strain. The F1 hybrid between the highly and poorly responding strains showed an intermediate secondary response. Since the highly responding strain showed a marked increase in PFC's during the secondary response, the primary course of immunization must have sensitized a large number of cells but induced antibody production in only a few. These sensitized cells were presumably then responsible 150

for the increase in the number of antibodyproducing cells following the second injection of antigen. In contrast, there was no such effect in the poorly responding strain, presumably because no large population of sensitized cells was available after primary immunization. We suggest, therefore, that the difference in the ability of the high and low responding strains to respond to immunization with poly(GluS2Lys33Tyr15) is due, at least in part, to the presence of more cells capable of being sensitized to the antigen in the highly responding strain.

Classes of immunoglobulin produced Radioimmunoelectrophoretic and Sephedex gel chromatographic studies were carried out to determine whether there were any differences in the classes of immunoglobulin molecules produced to poly(Glu52Lys33Tyr 15) by the highly and poorly responding strains 59'12o,122. The highly responding ACI strain produced antibody in all immunoglobulin classes studied (IgG1, IgG2, IgM), whereas antibody formed by the poorly responding strain was almost exclusively in the IgG class. These results suggest that an inability to make IgM antibody to poly(Glu s2Lys33Tyr 15) may be a factor in the poor response to that antigen. There was no correlation between the amount of antibody made and the immunoglobulin class.

Antibody affinity and specificity There are significant differences among the binding constants and specificities of IgG antibodies elicited by poly(Glu s ZLys33Tyr 15) in the highly and poorly responding strains 122. Using

60 v~ -< \

-4

20

ACZ AUG F344 •UF

AF

(4,4)(5,5)

(,,4) ([,4)(4,4)

(I,I} {6,6)

FA AF/AF AWAF AF/AF AF/F AF/F ACE F344 ([,4) H,r)

E[] > 5 0 0 ugAb/ml serum ] RAT [I~: I00- 499 ,ug Ab/ml ..... ] I < i00 ,ug Ab/rnl serum [

(I,4) it,I)

STRAINS

AND

ogg ogg Ag Ag

HYBRIDS

(genofype)

Fig. 6. The genetic control of the binding constants of antibodies to poly(Glu52Lys33TyP s) in various high responders, low responders and their hybrids. The quantities of antibody made by each group are keyed as indicated in the figure, and the Ag-B genotype of each group is shown in parentheses beneath each strain. Agg.Ag indicates those rats immunized with poly(GluS2Lys33TyP 5) aggregated with methylated bovine serum albumin. All others were immunized with unaggregated poly(Glu52Lys33TyP 5).

an equilibrium dialysis technique developed for studying the binding of macromolecules, high respopders were found to produce antibodies to poly(GluSZLys33TyP5) with higher binding constants than low responders (Fig. 6). The segregation of the binding constants in the F 1 and F 2 hybrids and in the backcrosses showed that the magnitude of the binding constant is under genetic control and is associated with the major histocompatibility locus of the rat. The ability to produce anti-poly(Glu52Lys33Tyr15) antibodies which crossreact with related synthetic polypeptides is also under genetic control (Fig. 7). This ability is inherited as a dominant trait and is associated with the major histocompatibility locus. Immunization of low responders with poly(Glu52Lys33TyP5) aggregated with methylated bovine serum albumin enhanced the quantity of antibody produced and led to the production of antibodies with higher affinity and greater crossreactivity. On the other hand, the antibody response in the high responders after immunization with aggregated antigen was decreased, and the antibodies had a lower binding constant and a lower crossreactivity.

The effect of adjuvants on the antibody response The use of adjuvants can greatly affect the magnitude of the antibody response to poly-

(Glu52Lys33Tyr 15) (Fig. 8) 124. The highly responding ACI strain made the most antibody following the use of complete Freund's adjuvant; therefore, maximal stimulation produced the maximal antibody response. In contrast, incomplete Freund's adjuvant was the most effective vehicle for immunization of the poorly responding F344 strain; thus, less intense antigenic stimulation elicited the maximal response. Further evidence supporting the necessity for moderate, sustained stimulation in eliciting a maximal antibody response lies in the greater effect of multiple (ten) injections of antigen in water compared to giving the same total dose of antigen in two injections in the poorly responding F344 strain. The genetically controlled differences in the antibody responses of the ACI and F344 strains were seen only following immunization with the antigen in complete Freund's adjuvant or with a primary and secondary dose of antigen in water. The effectiveness of the adjuvant in the ACI strain may be due to the slowness of antigen release and its prolonged effect on the cellular events occurring after stimulation. In the F344 strain, this maximal stimulation with adjuvant may have induced partial tolerance. When the _~ IOO P-. 75

[ ~

ACI (4,4)

F344 (I,I)

BUF (6,6)

A0

poly(Glu52Lys33Tyr 15 )

~7~ poly(Glu 56 Lys38Tyr 6) l

Ii FA AFIAF AFIAF ACT (1,4) (4,4) (1,4) agg

F344 agg.

Ag

RAT STRAINS AND HYBRIDS (genotype)

poly(Glu 57 Lys34phe 4)

Fig. 7. Crossreactivity between anti-poly(Glu s 2Lys 33Tyr 15) antibodies from various sources and related synthetic polypeptides, as determined by the precipitin reaction. The symbols are: [ZE, poly(GluS2Lys33TyPS); ~ , poly(Glu56Lys 38Tyr6); and I , poly(Glu57Lys34Phe9). The numbers in parentheses beneath each strain or hybrid signify the Ag-B type. Agg.Ag indicates those rats immunized with poly(Glu52Lys33TyP 5) aggregated with methylated bovine serum albumin. All others were immunized with unaggregated poly(Glu s 2Lys33Tyrl 5).

151

7°°I ki 6 0 0

F--] ACZ o~

cd

F344~

*1

200

~, I 0 0

CFA

+ Ag

rFA

Tbc

+

-I-

THEN

Ag

A(:J

Ag

CFA WATER WATER +

+

Ag

Ag

(MULTIPLE)

METHOD OF IMMUNIZATION

Fig. 8. The antibody response of the ACI and F344 strains to immunization with poly(Glu52Lys33Tyr 15) in various vehicles. The standard method of immunization was to administer the antigen in the appropriate vehicle divided among the hind footpads and the back of the neck. Three weeks after the initial immunization, the animals received antigen in water intraperitoneally and were bled 10 days later. The various vehicles containing the first injection of antigen were: (a) CFA, complete Freund's adjuvant containing additional tubercle bacilli (final concentration 3 mg/ml); (b) IFA, incomplete Freund's adjuvant; (c) Tbc + Ag, antigen mixed with 1.5 mg of tubercle bacilli; and (d) CFA then Ag, CFA followed in 5 days by antigen in water. The animals that received the antigen in water were immunized either with antigen divided into 10 daily doses with bleeding 12 days after the last dose (multiple injections) or according to the standard schedule described above.

antigen dissolved in water was given in two doses, it remained for only a brief period due to rapid degradation, and there was enough available to stimulate effectively only the highly responding ACI strain but not the poorly responding F344 strain. Conclusions

We have shown by several lines of evidence that the antibody response to poly(GluS2Lys33Tyr15) in the inbred rat is under polygenic control, and that the quantity, binding constant and specificity of the antibody vary concurrently and are most likely regulated by the same genetic control mechanism. A model for this genetic control mechanism is presented in Figure 9, and the major arguments for the model are outlined in Table 3. We propose that the R-gene controls the recognition of antigen, most probably by coding 152

for antigen receptors on the T-cell, and that this gene is linked to the major histocompatibility locus. By controlling one of the initial steps in the induction of the immune response, the R-gene precedes and can influence the expression of other genes in the system. The Q-gene is the major gene controlling the quantity of antibody produced, probably by influencing the number of specific B-cells in an animal capable of making antibody to poly(Glu s ZLys33Tyr 15). Other autosomal modifier genes, and possibly an Xlinked gene, also affect the quantity of antibody produced. Experimental evidence suggests that immunization with antigen aggregated with methylated bovine serum albumin partially corrects for some of the genetic defects in the low responder rats. It may overcome a defect in the R-gene function by allowing more efficient binding of antigen to the appropriate T-cells. Since the low responders immunized with aggregated antigen do not produce as high an antibody response as the highly responding strains immunized with poly(Glu52Lys33Tyrl 5) alone, other genetic defects in the low responder, probably among the modifier genes, are not overcome by immunization with aggregated antigen. The differences in the affinity and specificity of the antibodies produced by the high and low responders can reflect differences at several levels. The low responding strains could be deficient in the structural genes necessary to produce high affinity, crossreactive antibodies to poly(GluS2Lys33Tyr15). However, low responders immunized with the aggregated antigen can produce higher levels of antibody to poly(Glu 52Lys33Tyr15) which have a higher affinity and which are more crossreactive with related antigens. One would not expect that structural gene defects could be overcome by presentation of an antigen in the aggregated form, and this postulate suggests that the low responders possess the necessary structural genes for the production of high affinity, crossreactive antibodies, although there may be fewer B-cells with these genes. It seems more probable that the major immune HISTOC0 MPATIBIL~TY ALLELE COMPLEX

FI-DENE

Q-GENE

OTHER AUTOSOMAL MODIFIER GENES

[__]

X - LINKED GENE

[__]

LINKED

Fig. 9. Hypothetical model for the genetic control of the antibody response to poly(Glu52Lys 33TyrlS) in inbred rats.

Table 3 Summary of the evidence for each component of the polygenic model for the control of the antibody response in the inbred rat Component

Evidence Polygenic

Backcrosses and F 2 hybrids yie!d three populations of responders: low, moderate, and high.

References 59, 114

Two major autosomal genes (R and Q)

(1) When antibody responses were assigned to low, moderate, and high categories, the experimental ratios for the various hybrids agreed with a two gene model. (2) The fraction of extremely high or extremely low responders in the F 2 hybrid is that predicted by a two gene model. (3) When each possible genotype in the crosses between high and low responders is assigned a theoretical antibody response, the theoretical model fits the experimental model well. (4) Unexpectedly high or low antibody responses were found in all crosses in pedigree analyses and indicate that multiple genetic factors are involved. (5) Inbreeding of low responders yields low responders, inbreeding of high responders yields high responders and inbreeding of moderate responders results in the whole spectrum of antibody responses.

59,114

Linkage of one major gene to the major histocompatibility locus

(1) All representatives of each histocompatibility(Ag-B or H-l) group behave the same with respect to antibody formation whether they are low, moderate, or high responders. (2) Standard breeding studies demonstrate linkage of the antibody response to the Ag-B(H-1) antigen.

115,118

Other autosomalmodifier genes

Amounts of antibody made by rats with the same Ag-B(H-1) genotype, but having acquired the major histocompatibilityalleles by different breeding schemes, can be higher than the levels predicted by the two gene model, and these responses occur in both homozygotes and heterozygotes and in both males and females.

115

X-linked gene

(1) More high responders among females and more low responders among males. (2) The antibody response of the females is more heterogeneous in almost all strains and in their hybrids. (3) Physiological alterations in sex hormones do not affect the levels of antibody formed.

59,114,115, 117

r e s p o n s e genes c o n t r o l l i n g a n t i b o d y q u a n t i t y , affinity a n d specificity a r e o p e r a t i n g at the T-cell level, a n d studies in o t h e r systems have s h o w n t h a t T-cells p l a y a role in the p r o d u c t i o n of high affinity a n t i b o d i e s 12 s. If the low r e s p o n d e r s are deficient in the R-gene, which is p o s t u l a t e d to c o d e for the T-cell a n t i g e n receptors, insufficient a n t i g e n will b i n d to the T-cells to trigger t h e m to i n t e r a c t with the a p p r o p r i a t e B-cells. P r e s e n t a t i o n of the a n t i g e n in an a g g r e g a t e d form c o u l d facilitate the i n t e r a c t i o n of a n t i g e n with the Tcell, resulting in p r o p e r T-cell s t i m u l a t i o n a n d function. T h e m o d i f i e r gene c o m p l e x c o u l d be p l a y i n g a role in t h e p r o d u c t i o n of high affinity, crossreactive a n t i b o d i e s b y influencing the

n u m b e r of specific B-cells c a p a b l e of p r o d u c i n g such a n t i b o d i e s . T h e a n t i b o d y r e s p o n s e s to several o t h e r antigens are also u n d e r p o l y g e n i c c o n t r o l . In the m o u s e , the i m m u n e r e s p o n s e to sheep red b l o o d cells sT'ss, H-2.2 a n t i g e n specificity 32'33, thetaA K R a n t i g e n 36'37, YG2a a l l o t y p e 9°, the Yh i s t o c o m p a t a b i l i t y a n t i g e n 27 29, m o u s e Ea-1 b l o o d g r o u p antigens 43'44 a n d limiting doses of p o l y ( G l u 6 ° A l a 3 ° T y ' r l ° ) 126 a p p e a r to be g o v e r n e d b y m o r e t h a n a single gene, one o f which is u s u a l l y a s s o c i a t e d with the H - 2 locus. M o z E s a n d her colleagues 71'72 s h o w e d t h a t the a n t i b o d y r e s p o n s e in the m o u s e to the synthetic p o l y p e p t i d e p o l y ( P h e , G l u ) - p o l y ( P r o ) - - p o l y ( L y s ) is 153

governed by two genes: an H-2 linked gene controlling the response to the (Phe,Glu) portion of the polypeptide and a non-H-2-1inked gene controlling the response to the Pro--Lys portion of the molecule. In the human, MARSH e t al. 127 suggest that the immune response to a naturally occurring pollen antigen (Ra5) is governed by a histocompatibility-linked immune response gene in addition to a non-HL-A-linked recessive gene which controls the basal serum levels of IgE. There is support for the idea that some immune response genes may be associated with the Xchromosome. Studies by AMSBAUGHand her colleagues68,69 showed that the immune response to type III pneumococcal polysaccharide in the mouse is governed by an X-linked dominant gene in addition to other factors, presumably autosomal genes, that influence the magnitude of the response in mice possessing the X-linked gene. In man, several immune deficiency diseases are sex-linked 128-131, the X-chromosome carries genes that influence the serum levels of IgM 132-135 and neonatal differences in bacterial susceptibility occur when there are no significant differences in the levels of sex hormones x36-138. The control of the antibody response in inbred rats provides an interesting system in which to study the interaction of major and minor autosomal genes and of sex-linked genes in the regulation of one of the major mammalian host defense mechanisms. These studies can be extended to the cellular and molecular levels and may provide a fruitful approach to studying cellular differentiation and the genetic control of structure in mammalian systems.

Acknowledgement This work was supported by grants from the National Institutes of Health (AI 10611 and GM 00135) and from the Beaver County Cancer Society. We wish to thank Professor Samuel B. SALVIN and Dr. Donald V. CRAMERfor their critical review of this manuscript.

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