Antibody production by strain 2 and strain 13 guinea pigs

CELLULAR

IXIML’NOLOGY

Antibody

33, 245-256

Production

(1977)

by Strain

2 and Strain

I. Use of a Monomeric Guinea Pig Albumin Carrier Immune Response Gene Control 1

13 Guinea

Pigs

to Demonstrate

Homogeneous albumin has been isolated from the serum of strain 13 g-uinea pixs. The 2,4-dinitrophenyl (DNP) conjugate of this albumin (DKP, guinea pig albumin) can he used even at high doses (100 ,ug) to distinguish easily the nonresponder strain 2 guinea pigs from the responder strain 13 animals. This observation tnodifies the prcvious conclusion that clear-cut discrimination of responders and nonresponders requires the USC of low doses (i.e., 1 wg) of such a hapten-protein conjugate. Since albumin polymers as well as additional protein contaminants comprise a large proportion of some commercial albumin preparations, these ancillary molecules appear to be responsible for the previous suggestion that protein carriers differed from synthetic peptides in that low immunizing doses were required to distinguish responder from nonrespondrr animals. That responsiveness in nonresponders can be increased by inclusion of polgmerit forms of the antigen in the immunizing mixture raises the possibility that those cells nhich “process” antigens may play a major role in immune response (Ir) gene control.

INTRODUCTION Inbred 1Vright strain 2 and strain 13 guinea pigs can be used to demonstrate tile regulatory effects of histocompatibility-linked inmune response (Ir) genes (,l-3) In such studies, the general protocol followed is to select two different carriers, one which is highly iniiiiunogenic in both strains, the other which will elicit only a lmo~ responsein one strain. Both carriers are then conjugated lvith the samehapten and are used to imnmnize two similar groups of animals. one of each strain. Tlic antihapten responseinduced by the highly inmunogenic carrier is used as evidence that the sector of bone marrow-derived lymphocytes specific for this particular lqkn is functional in each strain. In contrast, animals inmunized with the second haptencarrier conjugate will produce a good anti-hapten responsein the responder animals but little or no responsein the second strain, the latter animals being referred to as nonresponders (l-3). Genetic studies can then be done to demonstrate linlcagr of * This investigation was supported by Grants R.R-07065-11 and AI-02158 from the 1r.S. Public Health Service. The latter grant was to M. W. Chase. z Abbreviations used : DNP, 2,4-dinitrophenyl ; DNFB, 2,4-dinitrofluorobenzenc ; BSA, bovine serum albumin; GPA, guinea pig albumin; HSA, human serum albumin; EEA, ethanol-extracted albumin; KLH, keyhold limpet hemocyanin; CF:Y, complete Freund’s adjuvant, TC.\, trichloroacetic acid.

Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0008

8740

246

ROBERT

T.

REESE

the ability to respond to histocompatibility genes (4-6, 10). The inability to give an anti-hapten response to the same hapten on the poorly immunogenic carrier is often related to some thymic lymphocyte-related phenomenon (4-10). In most studies, particularly in the more recent ones, synthetic polypeptides composed of a limited number of different L-amino acids have been used as carriers (7, 8, 10). Synthetic polymers have been chosen because of their relatively simple structures and because their generally poor immunogenic capacities allow some of them to be used even at high doses (100 pg) to distinguish responder and nonresponder animals unequivocally (7, 8, 10). Indeed, strain 2 guinea pigs will produce considerable amounts of anti-dinitrophenyl (DNP)Z antibody when stimulated with 100 pg or less of dinitrophenyl-poly-r.-lysine, when it is incorporated in complete Freund’s adjuvant (CFA) (9). In contrast, strain 13 guinea pigs produce little DNP-specific antibody when immunized in a similar fashion (9). This difference in carrier-determined responsiveness appears quite clear in such cases. Attempts have also been made to distinguish histocompatibility-linked Ir gene control on the basis of responsiveness to complex protein antigens (7, 8, 10). Many studies have concerned themselves with the use of proteins such as albumins as carriers (7, 8, 10). In these investigations, it has generally been necessary to use hapten-protein conjugates having relatively few hapten residues per molecule. This may be related to the fact that the fewer hapten residues present per molecule, the more native the conformation of the protein carrier and the more restricted the number of antigenic hapten-associated patches. What is not so clear is why limiting doses of antigen are necessary to demonstrate a distinct difference in responsiveness between responder and nonresponder animals when hapten-protein conjugates are used (7, 8). For example, in studies using antigens such as DNPr-BSA, pipsyls guinea pig albumin (GPA) and DNPB-GPA little difference was detectable between responder and nonresponder guinea pigs which had been immunized with any of the above antigens. Only when the immunizing dosage was reduced to 1.0 rg did unequivocal identification of nonresponder animals become possible ( 11, 12). The present investigation will demonstrate some of the factors important in one particular example of dose-related unresponsiveness to a purified hapten-protein conjugate. The example which has been studied is the responsive capacity of strain 2 and strain 13 guinea pigs to stimulation with 2,4-dinitrophenyl guinea pig albumin. The responsiveness to DNP-GPA has been compared to the responsive capacity of both of these animal strains when stimulated with the highly immunogenic molecule, 2,Cdinitrophenyl keyhole limpet hemocyanin (DNP-KLH) . It will be demonstrated that certain commercial preparations of guinea pig albumin contain several (at least six) other molecular forms in addition to monomeric albumin. It will also be shown that homogeneous guinea pig albumin can be prepared with ease, even though polymers may be generated during storage. Most important, evidence will be presented which suggests that, if the Ynonomeric form of DNPs-GPA is used, it is a very poor antigen in strain 2 guinea pigs and can be used even at high doses (100 pg) to distinguish nonresponder strain 2 guinea pigs from responder strain 13 animals. These data suggest that the major effect that reduction of antigen dosage may have is to decrease the concentration of contaminant molecules present in a preparation. It is these contaminants which may be responsible for the major immunogenicity of such albumin preparations when they are used to stimulate nonresponder animals.

IMMUNE

MATERIALS

RESPONSE

AND

REGULATION

247

METHODS

Preparation of guinea pig albunzin. The serum of strain 13 guinea pigs was diluted 1: 1 with water and then was fractionated twice with ammonium sulfate using the Adair and Robinson procedure as outlined by Kabat and Mayer ( 13). The partially purified albumin was dissolved in water, precipitated with an equal volume of 109; trichloroacetic acid (TCA), redissolved in water, and then extracted with 4 v01 of 95% ethanol at 4°C (14). The albumin was reprecipitated with TCA and was again extracted with ethanol. This product was dialyzed against water and freezedried. A 300-111g fraction of the ethanol-extracted albumin (EEA) was chromatographed on a 1.7 x 25.5cm column of DE-52 which had been equilibrated with 0.01 dl Tris-HCl, pH 80. The protein was eluted with a linear gradient formed from 400 ml of 0.01 &’ Tris-HCl, pH S.0, and 300 ml of 0.01 ill Tris-IICl. l)H 8.0, containing 0.3 M NaCl. OGcr puot& carriers. Commercial guinea pig albumin was obtained from Pentex (Miles, Kankakee, Ill.) ; keyhole limpet hemocyanin was purchased from Schwarz/ RIann (Orangeburg, N.Y.) ; and human serum albumin (HSA) was bought from 1Vortbington Biochemical Corp. (Freehold, N. J.) . Haptm conjuga.tion. Hapten-protein conjugates were prepared according to the procedure of Chase (15). Purified protein solutions ( -10 mg/ml) were allowed to react under alkaline conditions (pH 8.2 or above) with an alcoholic solution of 2.4-dinitrofluorobenzene (DNFB). The reaction was stopped by adding 1 vol of 203) glycylglycine, pH S.0, to conjugate with the unreacted DNFB. Unwanted byproducts were removed by dialysis. In this way, DXP&PA and DNP,,-IISA were prepared. Similarly, keyhole limpet hemacyanin was coupled with an average of five DSI’ groups per 68,000 daltons (DNP-KLH) so that the DYP-KLl-I I would have approximately the samedensity of DNP groups as did DNP&PA. Analytical procedures. Samples of guinea pig albumin were hydrolyzed as previously described (16). Amino acid analyses of the acid hydrolysates were performed by Dr. Glynn Wilson and Dr. Peter Blackburn of the Moore and Stein I,aboratory of Biochemistry at this University. Acrylamide disc gel electrophor&s was conducted as described by Davis and Ornstein (17). Protein-containing gels were stained with Coomassieblue (IS). Samples were subjected to immunoelectroplloresis in 17%agarose (Marine Colloids Inc., Rockland. Maine) using an ethylenediamineacetic acid, pH 8.9, buffer (19). The rabbit anti-guinea pig serum reagent used was capable of detecting at least 15 components in guinea pig serum. It was kindly supplied by Professor Merrill It’. Chase. Quantitative precipitin analyses were conducte(1 as described earlier (20). The amount of antigen precipitated was determined directly by its absorbance at 360 nm ; the total amount of protein precipitated was determined by absorbance at 278 mu or by the I,owry et al. [21 ) nlodification (22) of the Folio-Ciocalteu reaction. ~nrmmizations. Wright’s strain 2 and strain 13 guinea pigs were immunized wit11 the specific dosage of antigen emulsified in an equal volume of a 1: 1 mixture CJf complete and incomplete Freund’s adjuvant (Difco, Detroit, Mich.). A O.-l-ml volume of emulsion was distributed among the four footpads of each animal, Antigen binding assay. The ability of antibody to bind antigen was detected with a lnodification of the Farr assay (23) using 20-~1 aliquots of each of the antiserunl dilutions. After room-temperature incubation with 20 ,~l of a 1.77 x lo-7 .V solution of f-DSl’, a-succinyllysyl- [l’jI] iodotyrosine ( 14.9 mCi/mol), the mixture was

248

ROBERT

T.

REESE

FIG. 1. Immunoelectrophoretic analysis of guinea pig albumin. (A) Commercial guinea pig albumin (20 mg/ml) ; (B) ethanol-extracted guinea pig albumin (20 mg/ml) ; (C) DE-%?purified guinea pig albumin (22 mg/ml). Rabbit anti-guinea pig whole serum was used in each trough.

brought to 4°C and 457%saturation with ammonium sulfate which was also at 4°C. The ammonium sulfate precipitated the antibody-bound antigen, allowing free and bound antigen to be measured. KESULTS Albumin was separated from the serum of strain 13 guinea pigs by ammonium sulfate fractionation, followed by extraction with 95% ethanol. Some of the ethanolextracted albumin was further purified by chromatography on Whatman DE-52 as described above. The albumin preparations were either lyophilized (EEA), frozen, or sterile-filtered and refrigerated (DE-52 preparations) for storage. Before immunologic use, the state of purity of the two albumin preparations was compared to that of a standard commercial guinea pig albumin sample. Comparisons were made in two ways. First, concentrated solutions (20-22 mg/ml) of the albumin preparations were assessedby immunoelectrophoresis using a polyvalent rabbit antiguinea pig whole serum reagent. Photographs of the results are presented in Fig. 1. According to this criterion it is clear that the major molecular speciesin each of the preparations is guinea pig albumin. However, the commercial albumin contains at least two additional antigenic components of different electrophoretic mobilities, and EEA contains at least one extra antigenic species.To assessfurther the purity of these albumin preparations, approximately 100 pg of each was electrophoresed on a polyacrylamide disc gel. A photograph of the Coomassieblue-stained gels is presented in Fig. 2. From examination of the acrylamide gels it was found that the DE-52-prepared albumin contained predominantly monomeric albumin, a second higher molecular weight component, and a third very minor component which was

a

ethanol

b

c -

(a) 100 pg commercial; (b) 100 pg extracted; Cc) 100 pg DE-52 purifieci

FIG. 2. Acrylamide disc gels of electrophoresed guinea pig albumin preparations. (a) 100 pg of commercial GPA; (b) 100 pg of ethanol-extracted W.1; (c) 100 ,ug of DE-52-purified GI’A.

detected only when the stained gel was examined with a gel scanner. Integration under the peaks of the scanning profile suggested that this albumin preparation w;ts approximately 90% monomeric albumin. The EEA preparation contained larger amounts of the two higher molecular weight components plus a third additional component not observed in the DE-52 preparation. Examination of the gel containing the commercial albumin preparation revealed the presence of numerous other components (at least six) in addition to monomeric albumin. Furthermore, the COW taminants appeared to represent an appreciable proportion of the whole. Two other lots of commercial albumin were also examined. Acrylamitle disc gel electroplioresis of 1SO-pa e samplesfrom each of the three lots obtainetl at different times revealed Imt S to be somewhat less heterogeneous (apprositnately sis bantls)

sIrail

13

I)NI’,-CPA D&I’-KLII

5 100 100

0.0 f 0.28 2.i f 0.s.i 3.5 f 1.3

2.50

FIG.

DNPs-GPA NHIHCOI,

ROBERT

T.

REESE

3. Chromatography of DNP-GPA on Sephadex G-150. A 30-mg was chromatographed on a 1.6 X 82-cm column of Sephadex 0.050/o sodium azide.

sample (1.2 ml) of G-150 fine in 0.05 M

than the other two (Lots 7 and 465)) which were indistinguishable from one another (seven bands). Commercial albumin preparations, perhaps similar to the ones studied here, have been used in numerous biological experiments for years. Furthermore, lightly coupled commercial albumins have been used to define the responder status of strain 2 and strain 13 guinea pigs to DNP-conjugated protein carriers (11, 12). These earlier studies have suggested that, in order to detect a clear difference in the responder status of strain 2 and strain 13 guinea pigs to low coupled DNP-GPA, minimal doses of the DNP-protein are required for immunization (11, 12). Such results contrast with the observations that certain DNP-labeled synthetic amino acid polymers can be used to distinguish the responsiveness of these two guinea pig strains, even when high doses of immunogen are given (7-10). To test if this discrepancy is real or simply a reflection of the relative homogeneity of the preparations used, strain 2 and strain 13 guinea pigs were immunized with between 5 and 100 pg of DNPS-GPA obtained from the DE-52-purified albumin preparation. To demonstrate the responsive capacity of each of the guinea pig strains to strongly antigenie DNP-protein conjugates, other groups of animals were immunized with DNPKLH. The animals were bled 42 days after immunization, and the amount of antiDNP antibody in each serum sample was determined by quantitative precipitin analysis. The results of these analyses are given in Table 1. From the data it is clear that DNPS-GPA stimulates less than 20% as much anti-DNP antibody in strain 2 as in strain 13 animals. Furthermore, 100 pg of DNPQ-GPA only stimulated approximately 10% as much anti-DNP in strain 2 animals as could be stimulated in

IMMUNE

RESPONSE

2.51

REGULATION

similar animals with 100 pg of DNP-KLH. It should be pointed out at this time that these comparisons are not made at an early time when the amount of anti-DN P stimulated by 100 pg of DNPB-GPA in strain 2 animals is almost undetectable. Da! 42 sera were chosen because, by this time, the response of all groups of animals tested appeared to have reached a maximum. Indeed, comparison of the groups at an earlier time would undoubtedly have made the difference appear even more marked (24). Since we have clearly demonstrated that lightly coupled DNP-GPA is a poorly antigenic protein for stimulating anti-DNP antibody in strain 2 guinea pigs, it became more important to determine if the additional protein components observctl in the polyacrylamide disc gels of the DE-52-purified albumin preparation were albumin polymers or not. Therefore, 20 mg of DSPn-GPA were chromatocolumn of Sephadex G-l.50 fine, equilibrated in graphed on a I .6 x 82-cm 0.05 If N,HHCO,, OS/co sodium azide. The elution profile is shown in Fig. 3. Two major fractions were obtained, one colnprising approximately 939 of the total protein, the other about 7%. The contents of the two fractions \vere examined on polyacrylamide gels as before, with the results being presented in Fig. 4. Fraction 2 from the Sephadex column appeared to contain only monomeric guiiiea l)ig albumin. However, freezing and thawing of Fraction 2 protein enhancetl the fOrlll~~tiOll

Of the

1110kCUkX

fOrillS

fOUlld

in

~raCtiOl1

1 fro111

the

Se$dex

CO~~ltllll.

Furthermore, amino acid analysis of similarly fractionated unconjugated ( ; PA demonstrated that the protein of Fractions 1 and 2 had the same amino acid cornpositions (Table 2). Therefore, the protein in Fraction 1 is probably an albumin dimer. as indicated by its elution position from the G-150 column. Although not mentioned earlier, monomeric all)unlin can be obtained either 1)~.

(1) Fraction

I; (2)

Fraction

II.

FIG. 4. Acrylamide disc gel electrophoresis of DKP-guinea Sephadex G-1.50. (1) Fraction 1; (2) Fraction 2. These samples fore the Fraction 2 material was used for immunization.

pig albumin fractions were examined directly

from be-

252

ROBERT

T.

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TABLE Amino

Acid

Composition Amino

2

of Fractions acid

1 and

2 from

Fraction

Carboxymethylcysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine

1

Sephadex Fraction

21 49 26 24 76 25 19 53 29 2 1.5 52 20 23 14 48 18

G-150” 2

20 46 26 23 75 25 19 52 29 3 16 55 19 25 14 48 18

a The number of residues of each amino acid is based on a molecular of a residue were rounded off to the nearest whole number.

weight

of 68,000.

Fractions

ion-exchange or molecular-sieve chromatography of partially purified albumin. The problems arise in storage, as polymeric forms of the protein appear to be generated by lyophilization, freezing, or even maintenance in a sterile concentrated form at 4°C. An attempt was next made to demonstrate if monomeric DNPB-GPA is immunogenic for strain 2 guinea pigs. Freshly chromatographed (Sephadex G-150) DNPBGPA was incorporated into CFA, and the emulsion was injected immediately. In addition, some freshly chromatographed DNP9-GPA was held at 4°C for approximately 1 week. This material was examined by acrylamide disc gel electrophoresis and found to contain approximately 1% polymer. It was also incorporated into an emulsion with CFA and was used to immunize strain 2 guinea pigs. Sera were obtained as before, and the amount of anti-DNP produced was estimated by quantitative precipitin analysis. The data in Table 3 demonstrate that immunization TABLE l’recipitable

Anti-DNP Antibody Containing Varying Strain

No detectable polymer

Amount of precipitable anti-DPN antibody (w/ml)

3.3 f

of animals

indicated

Produced Amounts

to DNP-GI’A of Polymer

0.41

Preparations

Strain

13

Amount of polymer in DNP-GPA preparation

a Number

3

~107~

polymer

0.46 f

0.11

(4)a

in parentheses.

-1%

(8)

0.26

2

polymer

f

0.033

No detectable polymer

(3)

0.12

f

0.03

(3)

IMMUNE

I’crcentage -~

RESPONSE

of Hapten Bound by Varying Guinea Pigs Immunized with ~~ ~~

L?5,1

REClTI,A’I’JON

Amounts of Serum 100 pg of DNP-CPA

from Strain Monomera

2 and

I.3

Seru 111 (pl)

Hapten

Hapten

bound

by Strain (%I

bound

by Strain

1.00

0.66

0.20

0.10

85

8.1

67

33

23

1 .z

0

2

13

2

(5%) 1‘ Twenty microliters of n 1.77 X 10-T M solution mol) was used for the assay.

of c-l)SI’-l~~)l-[‘“jI]-iodo~~r~)sill~

(1-l.‘)

nl(‘i

of strain 13 guinea pigs with DNP&PA containing no detectable polymers induces no less antibody than found in similar animals immunized with DITP&I’A containing approximately 10% polymer, More important was the observation (1‘al)le 3) that, as the amount of polymer was reduced in the antigen used to stimulate strain 2 animals, there was a corresponding decrease in the amount of precipitable antibody detectable. In fact, the strain 2 animals immunized with D?\‘P&1’.4 containing no detectable polymer produced less than lcjc as much precipitable anti-DNI’ as similarly stimulated strain 13 guinea pigs. 7‘0 demonstrate that the precipitin assay provided an accurate estimate of the difference in the responses of strain 2 and strain 13 guinea pigs, sera from the two strains were examined for their abilities to bind a radiolabeled antigen. Although such binding assays provide only an estimate of the relative amounts of antihod! present, they are more sensitive than the precipitin assay and depend simply on antigen-antibody interaction. From Table 4 it is clear that the differences. which can be expressed in nGllig-rams, from the precipitin assay can also be detected in relative terms by the binding XS:ly. Thus, the strain 2 animals are not producing large amounts of anti-DNf’ antibody which n-as not detectable by the precipitin assay. In fact, the differences observed with the binding assay are equal to or greater than those seen by others (1 1, 12 ) n-hen they examined sera from strain 2 and strain 13 animals immunizetl wit11 lOO-fold less (1 ,IL~) DIYP-GPA than was used in these esl)eriments. These data confirm our previous suggestions that certain l)rotcin carriers ~111~ultl 11ot I>e classified as being less effective than pel)titles for tlistiiiguishing resl)ontl~7 ant1 nunresponder animals. TVhen guinea pig albumin which is honlogcneous is ei11ployed as a carrier, it can be used in a fashion similar tcJ pel)titles for distinguishing respondrr ant1 nonresponder animals. DISCUSSION In studies on the controls imposed by histocompatibility-linked Ir genes, synthetic polypeptides have generally been selected as carriers (2-10). A major reason for this selection is the observation that even those protein carriers having a relatively sniatl number of haptenic groups per molecule appear to require limiting doses ~II tmlCT

t0

tlelll~JllStrate

clearly

Ir

gelie

regdatOrj~

CUlltrd

(17-l

2).

In the 1)resent study it has been shown that D)N L’-labeled I/~OUO/J~CY~C all)ulllill \vill,

254

ROBERT

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REESE

if injected into strain 2 (nonresponder) guinea pigs in CFA, induce only low levels of DNP-specific antibody. This anti-DNP production was stimulated only if high doses of antigen (100 pg) were used. Furthermore, even at its peak around Day 42, the amount of anti-DNP antibody produced was quite small (less than 0.2 mg/ml) . In contrast, similar groups of strain 2 animals could produce almost 5.0 mg of antiDNP antibody/ml if stimulated with DNP-KLH (Table 1). The complementary data obtained from immunization of strain 13 guinea pigs with either DNP-GPA or DNP-KLH clearly demonstrate (Table 1) that, although DNP-GPA is a weaker antigen than DNP-KLH, responder guinea pigs immunized with large dosesof DNP-GPA (100 pg) produce approximately 80% as much antiDNP antibody as animals immunized with DNP-KLH (100 pg). Also, it is quite clear that, although the immunogenicity of the DNP-GPA preparation was appreciably reduced for strain 2 animals by removal of the polymerized albumin, the antigen was still quite capable of stimulating considerable anti-DNP antibody in strain 13 guinea pigs, even when they were stimulated with only 5 pg of antigen. Apparently, there is nothing exceptional about certain native protein carriers as compared with synthetic peptides requiring them to be considered apart with regard to the Ir gene regulatory controls which govern the induction of responsiveness. It is nonethelessdifficult under the experimental circumstances to determine, in an absolute sense,whether strain 2 guinea pigs can respond to haptenated monomeric albumin. Evidence has been presented which demonstrates that elimination of DNPR-GPA polymers from the DNP-GPA solution leads to synthesis of a decreased amount of anti-DNP. On the other hand, the possibility exists that emulsification of DNP9-GPA in CFA may induce somepolymer formation itself. Indeed, the hydrophobic environment of the emulsion might well shift the monomer-polymer equilibrium more in favor of the polymer, since the latter would have the smaller surface area. That is, a protein-protein environment might well be favored rather than an oil-protein environment. Thus, it is probably best to say that preparations containing predominantly the monomeric form of DNP-GPA, while being very poorly immunogenic in strain 2 guinea pigs, are quite immunogenic for strain 13 animals. From the observations of Grumet et al. (25) that thymic lymphocyte cooperation is not required for IgM production, it should not be surprising that some antibody is still produced by strain 2 nonresponder animals. The fact that large doses of monomeric DNPs-GPA trigger the production of lessthan 4% as much anti-DNP in nonresponders as in responders easily conforms to the ratio found for IgM and IgG in normal guinea pig serum (26). Studies are in progress to determine if the low-level anti-DNP production in nonresponders is IgM or someother isotype. In investigations by others it has been observed that clear-cut identification of nonresponder guinea pigs could not be made using lOO-pg doses of antigens such as DNPe-GPA and pipsy&,-GPA. Only when the immunizing dosage was reduced to 1.0 pg did unequivocal identification of nonresponder animals become possible (11, 12). The major importance of the present report is the realization that Ir gene regulation can clearly be demonstrated with even high doses (i.e., 100 pg) of hapten-protein conjugates, but only when they are in a homogeneousform. The commercial guinea pig albumin preparation which was examined was found to contain at least six proteins in addition to monomeric guinea pig albumin. The fact that most of the additional protein components were probably polymeric forms of albumin

IMMUNE

RESPONSE

255

REGULATION

is of little importance, since it was observed that polymerization of the albumin may appreciably increase its immunogenicity in strain 2 animals (Tables 1 and 3). Indeed, it has been known for years that polymeric forms of many proteins map be responsible for much, if not all, of their immunizing capacity (27-29). M’ith this in mind it is essential that, when commercially prepared proteins are obtained for immunologic purposes, they be used with proper reservations. Clearly, the ability tcb demonstrate obvious histocompatibility-linked Ir gene control over responses to natural protein antigens hinges on the purity of the protein preparations used. The fact that small doses of antigen have generally been required to display Ir gent regulation may simply mean that the protein preparations used have been contaminated with protein forms of considerable immunogenicity. Reduction of the protein dose may only have served to reduce the contaminants. That responsiveness in nonresponders can be increased by inclusion of polymeric forms of the antigen in the immunizing mixture raises the possibility that those cells which “process” antigens may play a major role in Ir gene control. Data supporting the hypothesis that the macrophage may be important in Ir gene control has recently been obtained by two other groups also (30, 31). Clearly, this must be studied in more detail. ACKNOLVLEDGMENTS Dr.

The author acknowledges M. W. Chase and Dr.

the technical T. P. King.

assistance

of Mr.

Gary

P. Saegel

and suggestions

frtrm

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Arquilla, E. R., and Finn, J., Science 142, 400, 1963. Pinchuck, P., and Maurer, P. H., J. Exp. Med. 122, 673, 1965. Kantor, F. S., Ojeda, A., and Benacerraf, B., J. Exj. &frd. 117, 5, 1963. McDevitt, H. O., and Tyan, M. L., J. Exp. Med. 128, 1, 1968. McDevitt, H. O., and Chinitz, A., S&m 163, 1207, 1969. Ellman, I,., Green, I., Martin, W. J., and Bcnacerraf, B., I’roc. X’at. rlcnd. Sri. 322, 1970. Control of Immune Responsiveness.” McDevitt, H. O., and Landry, M., “Genetic Press, New York, 1972. McDevitt, H. O., and Benacerraf, B., 1~ “Advances in Immunology” (F. J. Dixon Kunkel, Eds.), Vol. 11, pp. 31-74. Academic Press, New York, 1969. I.evine, B. B., Ojeda, A., and Benacerraf, B., J. Exp. Xcd. 118, 953, 1963. Gasser, D. I-., and Silvers, W. K., b “Advances in Immunology” (I~. J. Dixon Kunkel, Eds.), Vol. 18, pp. l-66. -4cademic Press, n’ew York. 1971. Green, I., and Benacerraf, B., J. Immunol. 107, 374, 1971. Green, I., Paul, W. E., and Benacerraf, B., J. Irnnzz~zol. 109, 457, 1972. Rabat, E., and Mayer, M., “Experimental Immunology,” 2nd Ed., p. 756. Charles Springfield, Ill., 1961. Kabat, E., and Mayer, M., “Experimental Immunology,” 2nd ed., p. 753. Charles Springfield, Ill., 1961. Chase, M. W., Personal communication, 1974. Keese, R. T., and Cebra, J. J., I~znzu~~toclzelrzisf~~l 13, 103, 1976. Davis, B. J., and Ornstein, L., In “Methods in Immunology and Immunochemistry” Williams and M. W. Chase, Eds.), Vol. 2, p. 38. Academic Press, Sew York, Spencer, M. E., and King, T. P., J. Biol. Chcm. 246, 201, 1971. Crowle, A. J., “Immunodiffusion.” Academic Press, Nebv York, 1961. Keese, K. T., and Cebra, J. J., J. I+r~mu~ol. 114, 863, 1975. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. B&J~. C&t. 1951.

U.S.-l

66,

Academic and H. G.

and

H.

G.

C Thomas, C Thnns,

(C. 196X.

:\.

193, 265,

256

ROBERT

T.

REESE

22. Chase, M. W., I.n “Methods in Immunology and Immunochemistry” (C. A. Williams and M. W. Chase, Eds.), Vol. 2, p. 273. Academic Press, New York, 1968. 23. Farr, R. S., 112 “Methods in Immunology and Immunochemistry” (C. A. Williams and M. W. Chase, Eds.), Vol. 3, p. 66. Academic Press, New York, 1971. 24. Reese, R. T., manuscript in preparation. 25. Grumet, F. C., Mitchell, G. F., and McDevitt, H. O., Anlz. N.Y. Acad. Sci. 190, 170, 1971. 26. Vaerman, J. B., and Heremans, J. F., J. Immunol. 108, 637, 1972. 27. Dresser, D. W., Immunology 4, 13, 1961. 28. Dietrick, F. M., and Weigle, W. O., J. Immunol. 92, 167, 1964. 29. Dresser, D. W., and Mitchison, N. A., In “Advances in Immunology” (F. J. Dixon and H. G. Kunkel, Eds.), Vol. 8, pp. 129-182. Academic Press, New York, 1968. 30. Greineder, D. K., Shevak, E. M., and Rosenthal, A. S., J. Immunol. 117, 1261,1976. 31. Miller, J. F. A. P., Vadas, M. A., Whitelaw, A., Gamble, J., and Bernard, C., submitted for publication.