GENETIC CONTROL OF SPECIFIC IMMUNE RESPONSES IN INBRED MICE*

F. Carl Grumet, Graham F. Mitchell and Hugh 0. McDevitt f

Division of Immunology, Department of Medicine Stanford University School of Medicine

Stanford, Calif. 94305

Genetic control of specific immune responses in inbred mice and inbred and random bred guinea pigs has now been reported for a wide variety of simple and complex antigens. For the most part, these genetic controls were discovered through the use of simple synthetic polypeptide antigens with a restricted range of antigenic heterogeneity, or through the use of immuniza- tion with very small amounts of natural proteins, in which case, presumably, one antigenic determinant becomes dominant with respect to immunogenicity. Many of these genetic controls have been shown to segregate as single, auto- soma1 dominant genes, and in the past two years a very large number of these genes have been shown to be linked to genes controlling the major (or, in some cases, the minor) histocompatibility antigens in both mice and guinea pigs. TABLE 1 is a brief and incomplete summary of those immune response genes which have been shown to be linked to the genes controlling histo- compatibility antigens.

As can be seen from the Table, the immune responses to a wide variety of synthetic polypeptide antigens, protein antigens, and at least one transplanta- tion antigen, are under an autosomal dominant genetic control which is linked to genes controlling the major histocompatibility antigens in mice and guinea pigs. The number and variety of these specific immune response genes, and the readiness with which they have been discovered in the past few years, sug- gest that there are many such genes affecting the immune response to a wide variety of antigenic determinants.

Our own studies have centered on the genetic control of the immune response to branched, multichain, synthetic polypeptides such as poly-(Tyr, G1u)-poly-D, L-Ala- -ply-L-Lys. As shown in TABLE 1, the ability to respond (T,G)-A--L is linked to the H-2b allele (and the H-2' allele derived by re- combination from the H-28 and H-2b alleles), and has been given the tentative designation of Immune Response-1 or Ir-1. For the sake of brevity, the major characteristics which have been described for Ir-1 are listed in TABLE 2.

The findings given in TABLE 2 indicate that we are dealing with antigen- specific genetic control over some step that is integral to the process of anti- body formation. The fact that nonresponders can be made to produce anti- (T,G)-A--L antibody when immunized with the antigen of a foreign carrier implies that both responder and nonresponder strains have the genetic information to produce antibody variable regions complementary to (T,G) -

*This research was supported by grants: from the United States Public Health Service, research grant A1 07757, and Special Research Fellowship A1 44178 to FCG; and from the American Cancer Society, California Division, research grant No. 505, and Dernham Junior Fellowship No. 162 to GFM.

t Senior Investigator of the Arthritis Foundation.

170

Grumet et al.: Immune Responses in Inbred Mice 171

TABLE 1

HISTOCOMPAT~BILITY LINKED GENETIC CONTROU OF SPECIFIC IMMUNE RESPONSES

Antigen Species Linkage Reference

1. (T,G)-A- -L 2. (H,G)-A- -L 3. (Phe,G)-A- -L 4. Bovine gamma globulin (low dose) 5 . Ovomucoit(1ow dose) 6. Ovalbumin (low dose) 7. Mouse erythrocyte antigen Ea-1a.b 8. Mouse male (Y) transplantation

9. Hapten-poly-L-lysine antigen

10. Bovine serum albumin (low dose)

11. Human serum albumin (low dose)

12. G,A

13. G,T

14. Tri-nitro-phenyl hapten

Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse

Guinea pig

Guinea pig

Guinea pig

Guinea pig

Guinea pig

Mouse

H-2b. i H-28, k . 11

H-2as.b.d.h,i. k .q

H-28. k H-P’.k H-2b .d .n

H-2b . i H-3 or H-6

Strain 2 histocom- patibility antigen

Strain 2 histocom- patibility antigen

Strain 2 histocom- patibility antigen

Strain 2 histocom- patibility antigen

Strain 13 histocom- patibility antigen

H-2’

1 1 1 2 2 3 4, 5 6 8

9, 10

11

12

13

13

14

A- -L, but that the nonresponder strain lacks the ability to recognize (T,G)- A- -L as a foreign immunogen. Although the evidence is not yet final, all the available data indicate that the antibody produced in this way in nonresponders has the same specificity as the antibody produced by responder animals. Since Ir-1 is not linked to the Ig immunoglobulin allotype locus,1G and since variable region genes and constant region genes appear to be linked in the rabbit, there is very little reason to think that Ir-1 has anything to do with genes coding for the structure of heavy-chain variable regions.

Given the supposition that the Ir-1 gene affects the recognition of immuno- genicity by a cell other than a precursor of the antibody producing cell itself, and the fact that this type of immune response gene affects delayed sensitivity and graft rejection as well as antibody formation, it is reasonable to postulate that the Ir-1 gene is in some way involved in the genetic control of antigen recognition at the level of thymus-derived, antigen-reactive cells. The close and frequent association of immune response genes with genes governing major (and minor) histocompatibility antigens suggests either that the Ir-1 genes are a separate antigen recognition system operative in thymus-derived cells, or that histocompatibility antigens have a marked effect on the interac- tion of antigen with immunoglobulin receptors on the surface of thymus- derived cells. Some evidence on this point can be gained by precise mapping of the many immune response genes, and these mapping studies are currently underway in several laboratories. Effects due to histocompatibility antigens themselves would result in immune response genes mapping at many different

172 Annals New York Academy of Sciences

TABLE 2

CHARACI'ERISTICS OF THE IR-1 GENE

1. Ir-1 is an autosomal dominant gene with a quantitative effect on the amount of antibody produced to antigens such as (T,G)-A- -L. The various alleles of this gene are specific for the amino acid composition of the antigenic determinant (e.g., (T,G)-A- -L, (H,G)- A- -L, (Phe,G)-A- -L).

2. The Ir-1 gene maps in the center of the H-2 complex, just to the right of the Ss (serum substance) gene and just to the left of the k region of the H-2 complex.'6

3. The immune responsiveness controlled by the Ir-1 gene is transferable with immuno- competent cells, e.g., spleen cells, fetal liver cells, or purified peripheral blood lymphocytes.'

4. The responder allele of the Ir-1 gene(s), when present, affects not only the amount but the specificity of the antibody produced.'

5. The major difference between responder (high responder) and nonresponder (low responder) strains of mice is not in the primary response, but is in the secondary response to aqueous antigen injection.16

6. The difference between responder and nonresponder strains can be completely abrogated by immunization and boosting with (T,G)-A- -L complexed with an immunologically recognizable, foreign, charged albumin, such as methylated bovine serum albumin.16

7. The ability to respond well to (T,G)-A- -L is a thymusdependent immune response."

points throughout the H-2 region. On the other hand, if there is such a thing as a separate antigen recognition system, it is conceivable that it would map in an antigenically silent (with respect to transplantation rejection) region either within or to either side of the H-2 region.

Direct proof that the Ir-1 gene is expressed in thymusderived cells is not yet available. Attempts to obtain such proof through the use of radiation- induced chimeras produced by the injection of responder fetal liver cells into irradiated nonresponder recipients met with a number of technical difficulties, and we are currently carrying out these studies in tetraparental (allophenic) mice.*s One of the technical difficulties involved in immune response genes linked to histocompatibility antigens is that cell transfer studies are almost always complicated by host versus graft or graft versus host reactions. These are particularly troublesome in our standard immunization program, which utilizes immunization in Freund's adjuvant followed by a three-week waiting period before a secondary injection of antigen in aqueous solution. To avoid the complicating effects of Freund's adjuvant, as well as t o compare a pure primary response to (T,G)-A- -L in responder and nonresponder animals, we have recently studied the immune response to aqueous injections of (T,G)- A- -L in responder (C3H.SW) and nonresponder (C3H/HeJ) mice. The initial results indicated that responses to aqueous injections of (T,G)-A- -L could be readily detected readily by increasing the sensitivity of our standard antigen-binding assay. To do this, the specific activity of (T,G)-A- -L-I lz6 is

Grumet et al.: Immune Responses in Inbred Mice 173

increased, and the ratio of antiserum to antigen is increased fifty to one hun- dred fold above that used in the standard antigen-binding capacity assay.ln

Doses ranging from 0.001-100 pg of antigen were used. The lower doses resulted in nearly undetectable responses, while the responses at 10 pg and 100 pg of antigen given as primary and a secondary antigenic stimulus in aqueous solution were essentially similar. Only the results of immunization with 100 pg (T,G)-A--L in aqueous solution are given in FIGURE 1 and 2. In FIGURE 1, the responses to 100 pg of (T,G)-A--L in aqueous solution on day 0, day 8, and day 30 are shown for C3H.SW and C3H/HeJ mice. Both strains gave an equal early primary response which peaked at about four days with the production of a small amount of almost exclusively 19s (by 2-mer- captoethanol sensitivity, and chromatography on Sephadex G-200) antibody. Thus, the primary response to (T,G)-A--L appeared to be equal in the two strains, and this early 19s primary response has recently been shown to be radiation-sensitive. The response to a second injection of aqueous antigen eight days after the primary injection resulted in a marked difference between the responder and nonresponder strains. The responder (C3H.SW) strain pro- duced a rapid and marked 7s (Zmercaptoethanol resistant) secondary anti- body response, while the nonrespnder (C3H) mice produced neither a 7s secondary antibody response nor a repeat 19s primary antibody response.

FIGURE 1. Responses of C3H.SW and C3H/HEJ mice to lOOpg of (T,G)-A-L in aqueous solution. Key: C3H.SW total antibody-.-; C3H.SW 7s antibody - - -0- - -; C3H total antibody -O-; C3H 7s antibody - - -0- - -.

174 Annals New York Academy of Sciences

Although these results are similar to those obtained earlier with immunization with (T,G)-A--L in Freund’s adjuvant,lG the use of antigen in aqueous solu- tion has permitted a much clearer demonstration of the difference in the secondary response. to (T,G)-A- -L in responder and nonresponder strains.

I- L E ” w 0 20

10

0 2

DAYS AFTER FIRST INJECTION

70 4 n

2 m 601

loop0

1 I

I

w n 20-

DAYS AFTER FIRST INJECTION

FIGURE 2. Antibody response of thymectomized and Sham-thymectomized non- responder (C3H/HeJ) and responder (C3H.SW) mice. Key: Thymectomized total antibody -0-; Thymectomized 7s antibody - - -0- - -; Sham-thymectomized total antibody -0-; Sham-thymectomized 7s antibody - - -0- - -.

Grumet et al.: Immune Responses in Inbred Mice 175

If the Ir-1 gene is, in fact, expressed in thymus-derived, antigen-reactive cells, a minimum prediction of this hypothesis would require that thymecto- mized responder mice would respond to this type of immunization regimen in a manner similar to normal or thymectomized nonresponder mice. While such a result would not prove that the Ir-1 gene is expressed in thymus-derived cells, any other result would argue against this hypothesis.

In FIGURE 2, the antibody response of thymectomized and shamthymecto- mized nonresponder (C3H/HeJ) .and responder (C3H.SW) mice is shown. All of the mice in these experiments were either thymectomized or sham-thy- mectomized, given a lethal dose of irradiation, and protected with syngeneic bone marrow cells. They were immunized four to five weeks following thy- mectomy, irradiation and cell transfer. As is shown in FIGURE 2, the nonre- sponder mice give the same type of early 19s primary antibody response, and fail to give any response to a secondary or tertiary injection of aqueous anti- gen. On the other hand, thymectomized responder (C3H.SW) mice appear to respond in a manner nearly identical to the nonresponder mice, with an early, weak 19s primary antibody response and no secondary or tertiary antibody response of either 19.9 or 7s type. The sham-thymectomized responder mice give a 19s primary and a rapid, marked 7s secondary and tertiary antibody response, as do normal responder mice.

These results are thus compatible with, although they do not prove, the hypothesis that the Ir-1 gene is expressed in a thymus-derived, antigen-reactive cell type. Direct proof of this hypothesis will require the conversion of a non- responder into a responder animal by the transfer of thymus-derived cells. Such experiments have failed in the past, at least in part because of the pro- longed immunization time and the histoincompatibility problems inherent in this system. The use of aqueous antigen, combined with a shorter period of immunization, is a more favorable experimental situation in which to transfer either normal or “educated” thymus-derived cells from responders into thy- mectomized, lethally-irradiated, bone marrow-protected nonresponder re- cipients.

These results also suggest that one of the major functions of thymus- derived, antigen-reactive cells is to facilitate the development of a 7s second- ary antibody response. Similar results, in which thymectomy has a greater depressive effect on the 7s secondary response than it does on the 19s primary response have also been reported by *l The question of whether this is a general phenomenon and a major part of thymic function in this or in other systems will require further experimentation, particularly transfer studies using normal and “educated” thymus-derived lymphocytes.

References

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176 Annals New York Academy of Sciences

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Grumet et al.: Immune Responses in Inbred Mice 177

DISCUSSION

DR. Corn: I would like to know if you’re willing to interpret your results to mean that the thymus, the special thymus recognition system, which you’ve proposed is not required for the 19s response but is only required for the 7s response?

DR. MCDEVITT: I think in this system it would appear that way. In other words, it may very well be like the pneumococcal polysaccharide system which is also thymus-independent and predominantly a 19s response. Further, this is also a polyvalent antigen like pneumoccoccal polysaccharide.