problem and paradigms: i–j: a rogue's riddle

278 BioEssays Vol. 4, No. 6

Problems and Paradigms presents discussions of fundamental biological problems and of phenomena whose basis is not clear within the present framework of ideas. Below are presented two views of the puzzle of the I-J determinants.

I-J: A Rogue's Riddle Colleen E. Hayes

'My Dear Mr Holmes, I should be very glad of your immediate assistance in what promises to be a most remarkable case. ' Sir A. Conan Doyle, 1904.

Introduction

An obscure locus encoding a vaguely characterized protein product agitated immunologists when it disappeared from the established genetic map. This notorious gene is provoking us once again. Its disquieting disappearance may relate to issues at the heart of immunobiology. However, the story must begin with the immune system itself.

Infectious microorganisms provide a perilous environment for us. Our immune system spares us from inevitable death by infection. It must discriminate antigens from self-molecules, then eliminate the antigens. The term antigen refers to foreign molecules, the term self to our body's own molecules.

The T lymphocytes distinguish foreign antigens from self-molecules and then orchestrate an appropriate immune response. The helper/inducer T cells begin the response and the suppressor T cells end it, directing the immune system as a conductor might direct an orchestra.

Regulatory T cells, like conductors, are selective. A response to one antigen can be initiated while the response to another antigen is terminated: violins may be signaled to play, trumpets to remain silent. A decade's experimenta- tion has established a strong immuno- logical paradigm. Immune T lymphocytes cannot recognize foreign antigens out of the context of self. Rather, they recognize a combination of antigen and certain self-proteins displayed together on macrophages. Conductors cannot interpret dots on a page; the dots become musical notes only in the context of a musical clef.

When regulatory T lymphocytes fail, random, discordant sounds replace the music. Without helper/inducer T celI function, responses don't begin. This is immunodeficiency. Acquired immune

deficiency syndrome is a well-publicized example of helper/inducer T cell failure. Without suppressor T cell function, responses don't terminate, or inappro- priate responses begin. Immunity may be erroneously directed to oneself; this is autoimmunity. Together, helper/inducer and suppressor T lymphocytes produce a harmonious result.

A surge of biochemical and motecular genetic evidence has unlocked some secrets of helper/inducer T lymphocytes and their antigen-binding receptor pr0teins.l The suppressor Tlymphocytes pose a formidable challenge to immuno- biologists; there is as yet no clue to their recognition structures. Indeed, research on the only structure that characterizes these cells, the I-J determinant in mice, produced more questions than answers.

Some evidence suggests that the murine I-J determinant may reside on a suppressor cell protein that discriminates between self and foreign molecules.* Unraveling the tangled genetic and biochemical analyses done on I-J determinants may solve some mysteries of suppressor T cell function. When we can describe molecules used by suppressor T cells in detail, we may begin to understand how regulatory T cells orchestrate a harmonious immune response. This article focuses on the xnresolved puzzles relating to the I-J determinant.

I 4 Determinants are the Hallmarks of Suppressor T Cells A cascade of events produces immunosuppression. The k s t type of suppressor T cell responds to an antigenic stimulus by secreting a small, biologically active protein (lymphokine). The lymphokine binds to T cells of a second type, inducing them to become suppressive, and amplifying the original signal. Some researchers describe a third T cell in the sequence. Suppressor T cell immunobiology was reviewed re~ently.~

The I-J determinant was defined immunochemically in mice.4 The cells and proteins involved in the suppressive

cascade display determinants (epitopes) that bind to certain antibodies. Anti- serum to these I-J determinants can be produced in several ways; five labora- tories isolated monoclonal antibodies to I-J determinankg

Mouse strains are classified according to the I-J determinants displayed by their suppressor T cells and suppressive lymphokine~.~ Antiserum produced by immunizing strain BlO.A(3R) mice with strain BlO.A(5R) T cells immunochemically distinguishes the I-Jk determinants. Similarly, antiserum produced by immunizing strain BlO.A(SR)mice with strain BlO.A(3R) T cells distinguishes the I-Jb determinants. The assays for I-J determinants depend on these antisera, or monoclonal antibodies, and are often difficult to

Biochemical Details on I-J Molecules are Scanty

There is copious information on suppressor T cell biology,8 but meager biochemical data on proteins with I-J determinants (Table I). Molecular weight estimates vary from 15,000 to 60,000 dalton~.~- '~ Progressive technical refinements account for some variability, but recent, relatively precise analyses still vary from 15,000 to 35,000 daltons.5-', lo, Is

Some I-J molecules are monomeric, others are heterodimers (Table I). An antigen-binding site occurs on some polypeptides carrying the I-J epitope, but not on others. Most authors describe an immunosuppressive biological function, but one report concerns a protein that inhibits phospholipase and N- linked glycosylation of an IgE-binding lymph~kine.~ If the data summarized in Table I are accurate, then several distinct molecules bind to I-J-specific antibodies.

The I-J determinant may be a protein, an oligosaccharide, or some other structure . Alpha-mannosidase digestion was found to destroy the I-J determinant ;l* the effect was dependent onenzyme concentration and was specifi- cally inhibited by mannose analogues.

BioEssays Vol. 4, No. 6 279

PROBLEMS AND PARADIGMS

TABLE I. Characteristics of proteins with I-J epitopes

Mr Subunit structure

~

Antigen- binding Function (antigeny Ref.

15,Ooo

19,000- 30,000

25,000 25,000

25,000- 28,000

25,000- 28,000

33,000- 68,000

35,000- 55,000

40,000- 50,000

50,000-

66,000

Single polypeptide

Single polypeptide;

Single polypeptide Non-covalently associated with M, 85,000 antigen- binding protein to give M , 200,000 molecule

DisuUide-bonded to M, 45,000 antigen-binding protein

Noncovalently associated with M, antigen-binding protein

n.d.

n.d.

forms homodimers

n.d.

Single polypeptide

Heterodimer ; M, 35,000 and 42,000 subunits

No

YeS

No No

No

No

Yes

Yes

Yes

Yes

Yes

Inhibits phospholipase and N-linked oligosaccharide assembly to IgE binding lymphokine

TsF, (GAT)

Cell surface (NP) TsF, (SRBC)

Secreted TsF (KLH)

Extracted TsF (KLH)

TsF, (ABA)

TsF, (KLH)

TsF, (GAT)

TsF, (KLH)

TsF, (GAT)

5

6

7 8

10

11

12

13

14

15

a Antigens: GAT, glutamic acid, alanine, tyrosine copolymer; Np, nitrophenol; SRBC, sheep red blood cells; KLH, keyhole limpet hemocyanin; ABA, azobenzenearsonate; n.d., not determined.

Other investigators have not tested the chemical nature of I-J determinants in their systems.

Some important biochemical characteristics have not been reported for proteins with I-J epitopes. A published amino acid analysis must be reviewed cautiously, since a protein mixture was analyzed.6 Other researchers have reported neither amino acid composition nor sequence analysis. The isoelectric points of proteins listed in Table I are unknown. Ascribing suppressive function to a well-characterized protein and elucidating thechemical nature of the I-J epitope are the first I-J dilemmas that must be resolved

The Case of the Misplaced Locus

To locate the structural gene encoding the suppressive lymphokine with the I-J determinant, researchers analyzed two strains, BIO.A(3R) and B.lO.A(SR) (hereafter 3R and 5R; ref. 4). These strains serve as prototypes; the 3R strain produces suppressive proteins with I-Jb epitopes, the 5R strain's suppressive proteins have I-Jk determinants. The 3R and 5R strains are genetically very similar; they derive from crosses made three decades ago.

Immunogeneticists interested in the H-2 gene cluster on mouse chromosome

17 bred strains that were genetically identical except in their H-2 genotype." An A/WySn x C57BL/10 hybrid was backcrossed to its C57BL/10 parent strain. Selected H-2-heterozygous off- spring were backcrossed again to the C57BL/ 10 strain. This breeding scheme yielded the BIO.A strain after twenty backcross generations and one intercross to give H-2 homozygotes; BIO.A retains

only the H-2 genes from A/WySn (Table 11).

The 3R recombinant was discovered at backcross generation seven and the 5R recombinant at backcross generation eleven." Each recombinant strain de- rives the left half of H-2 from its C57BL/10 parent and the right half from its A/WySn parent (Fig. 1 ; Table 11). Those who discovered the suppressive lymphokine's I-J epitope concluded that the intra-H-2 recombination points in 3R and 5R must bracket a length of chromosome with the lymphokine's structural gene, Z U - ~ . ~

The 3R and C57BL/10 strains were designated I u - ~ ~ , the 5R and A/WySn strains I u - ~ ~ (ref. 4). The genetic analysis presumed that the 3R and 5R strains have alleles at Za-4, the same number and position of loci, single, homologous recombinations, no muta- tions, and identical genes elsewhere. Abundant experimental evidence supported this analy~is.~.

Molecular biologists later identified several genes in H-2 and discovered that the 3R and 5R crossovers (and many others) occurred within a 4 kb interval inside the Ea gene (Fig. 1 ; ref. 18). Four kb is too small to encompass a typical eukaryotic gene. The DNA probes spanning this interval, 60 kb to the left, and 100 kb to the right, did not hybridize with suppressor hybridoma T-cellmRNA at a sensitivity of 3 mRNA molecules/cell.lB No DNA rearrange- ment involving this 4 kb region was detected. Hybridoma T cells apparently do not transcribe Ag, A,, Ep E and E,, nor do they splice an exon fikated

TABLE II. Genetic composition and I-J phenotype. of several strains

Genotype

H-2 loci - - -

I-J Strain K Ap A, Ej E, Slp C4 D Background phenotype

A/WySn k k k k k d d d a C57BL/10 b b b b b b b b b B1O.A k k k k k d d d b A.BY b b b b b b b b a BI0.A(2R)a k k k k k d d b b BIO.A(4R)B k k k k / b b b b b b B 1 O.A(3R)B b b b b / k k d d d b* B 1 O.A(SRY b b b b / k k d d d b 3RxC57BL/lO b b b b/k k d d d b/k

b b b b b b b b b 3R x A.BY b b b b/k k d d d b/k

b b b b b b b b a

k b k b k b b k k (h also?) b

* The BIO.A(2R), BIO.A(3R), BlO.A(4R), and BIO.A(SR) recombinants were detected during the backcrossing of the A/WySn H-2'L loci on to the C57BL/10 background." The BtO.A(3R), BlO.A(4R) and BIO.A(SR) recombination points are localized within the Eb gene; BlO.A(ZR) recombined between C4 and D (Fig. 1). As explained in the text, the BIO.A(3R) (b*) and C57BL/10 (b) background genes are not equivalent.

280 BioEssuys Vol. 4, No. 6


Recombination events

H BlO.A(BR) B 1 O.A(4R) 61 O.A(5R)

Scale I I I 1 I I I I I I I I I I 1 I 1 I L J J I I I I I I I I I I I I I I I I I 1 I I I I I I I I I I I 1 I I 1 I I I I I I I I I I I I I I I 1 I I I I I 1 I I I I I I 1 1 I I I I 1 1 I I I I 1 I I 1 Id kb 0 100 200 300 400 500 600

Fig. 1. Genetic loci in ihe mouse chromosome 17 H-2 complex. ( I B r 33)

within the 4 kb interval to exons encoded elsewhere during mRNA pro~essing.~~

In one disquieting motion, molecular genetic analysis swept the Ia-4 locus from the H-2 map. Those studying immunosuppression were left to explain not only where the Ia-4 gene really is, but also why it appears to be H-2-associated.

‘Well, Watson, what do you make of it?’

Single Gene Models are Now lmplausi ble

No single gene model placing Zu-4 anywhere in H-2 is plausible. Positioning Iu-4 centromeric to the recombination hotspot in the Ep gene would require 5R to be a triple recombinant. The standard recombination frequency between Ep and K is < 0.12% ; two independent recombinations between these genes would occur at a frequency of c 0.01 % . Positioning Iu-4 telomeric to the recombination hotspot would require 3R to be a triple recombinant. The standard recombination frequency between Ep andDis < 0.37% ;heretwoindependent crossovers would occur at a frequency of < 0.14%.

A mutation yielding a short 6-type sequence 3‘ to Ep in 3R would resemble a triple recombination. Though unlikely, such a mutation has not been ruled out by actual DNA sequence comparisons. Indeed, several authors propose that H-2 polymorphisms result from a mutational process that transfers small blocks of nucleotides among members of a multi- gene family, the recipient gene mutating several nucleotides under the influence of an unmodified donor gene.2o

Accounting for I-J immunochemical polymorphisms using a regulatory element in the 4 kb interval and the Za-4

locus elsewhere in H-2 is very unlikely. The h y p e element in strain 3R would have toenhance transcriptionofanZ~4~ locus, and the k-type element in strain 5R would have to enhance transcription of 1~2-4~. There is no precedent for such a regulatory element, and positioning Iu-4 would still be difficult.

A single gene model placing Iu-4 outside H-2 is inconsistent with the available data. In the nonrecombinant, homozygous strains, the H-2 genotype, not the background (genes excluding H-2) genotype, predicts the observed I-J determinant phenotype (Table 11). Strain 3R excepted, the E, genotype accurately predicts the I-J phenotype of recombinant strains.

Two Gene Models are also Problematic

A key result in unraveling the Za-4 locus puzzle came from two hybrid crosses 3R x C57BL/10 and 3R x A.BY (Table 11). The first hybrid made proteins with the I-Jk determinant, the second hybrid did not.3921 Evidently at least two genes control I-J determinants; one of these is in the 3R’s H-2 complex but not in H-Zb, the other is somewhere in the C57BL/10 background. The 3R genotype at the presumed background locus is not equivalent to C57BL/10; rather, it must derive from A/WySn. The probability of retaining an unselected A/WySn gene in the 3R background after ten backcrosses to C57BL/10 is just 0.20%. Nevertheless one assump- tion made in the original genetic analysis appears to be incorrect.

A simple two-gene model for controlling I-J determinants might use one H-2 locus (perhaps E,) and one background locus. However, such a model does not accurately predict the I-Jb phenotype of strain 3R sumressive Droteins. Since 3R

700 800 900 1000

has the H-2k gene required for I-Jk determinants, its suppressive proteins should resemble 5R if it has a C57BL/ 10 gene at the second postulated locus and A/WySn if it has an A gene there. Instead, 3R proteins resemble C57BL/ 10’s suppressive lymphokines.

The 3R’s I-Jb determinants cannot be explained using a two-gene model. At least three genes and probably more are required to explain the data (Table II).The fact that H-2k strains have a functional E, gene, whereas H-2b strains have a nonfunctional E, gene is almost certainly relevant to the problem. It is also possible that I-Jb and I-Jk determinants are not actually products of alleles. Untangling the genetic snarl is the next step in solving this puzzling case.

I 4 Epitopes and T Cell Receptors

As described above, regulatory T lymphocytes distinguish self from non-self. They do so by using certain self-proteins as reference points to form a context in which an antigen is recognized.2e The T cell’s plasma membrane receptor protein is envisioned as having a complex binding site with one portion accommodating an antigen fragment, and another portion accommodating a self- protein.’Occupancy oftheentirebinding site is thought to activate the T cell. The noncovalent complex of an antigen fragment and a self-protein forms on the macrophage plasma membrane.

The helper/inducer T cell’s receptor protein exhibits remarkable plasticity. Alternative antigen-binding sites seem to result from varied gene rearrange- ments in a manner similar to the immunoglobu1ins.l Self-specificity is also malleable, but not well understood. Precursor cells forced to differentiate in an alien environment vield a no01 of

I I

mature T cells that recognize the alien self-proteins in preference to their own. Although the receptor structure that accounts for self-specificity is unknown, it is likely to be a portion of the identified T-cell receptor heterodimer,' or to be a protein derived from similarly rearranging genetic elements.

The A p A,, Eg, and E, genes encode the self-proteins used as reference points by helper/inducer T cells (Fig. 1; ref. 22). Although these genes are not actually transcribed in regulatory T cells, they probably exert a selective influence on the maturing T-cell pool. The differentiating T cells that acquire appropriate receptors for these self- proteins are presumably selected; those without sui table bindingsi tesarepresum- ably terminated. This differentiation and selection process probably occurs in the thymus. Schrader theorized that some T-cell receptor proteins would reflect H-2 polymorphisms, although they would not be H-2-en~oded.~~

To account for the fact that non- transcribed H-2 loci seem to control I-J determinants, Tada proposed that I-J determinants reside on the suppressor T cell's receptor for self-proteins.2 We suggested that E, is probably the controlling H-2 ~OCUS, '~ and provided evidence that I-J-specific antibodies partially block the cellular interaction between macrophages displaying antigen fragments and immune T lympho- c y t e ~ . ~ ~ Other data are also consistent with Tada's hyp~thesis.~ However, some evidence is inconsistent with the postulated receptor function for proteins with I-J epitopes.

I4 Determinants on Macrophage Plasma Membranes

The occurrence of I-J determinants on macrophages challenges the receptor hypothesis. Three groups coated macrophages with I-J antiserum, washed them, and tested their function; the antibodies blocked the macrophage's ability to stimulate an immune res- p ~ n s e . ~ ~ - ~ ' These researchers suggest that I-J determinants occur on macrophage proteins, where they provide the context for suppressor T cells to recognize antigen. The antiserum probably contains multiple antibody specificities, so these studies must be repeated with monoclonal antibodies.

Perhaps the macrophage membrane can passively acquire the proteins with I-J determinants. We analyzed macrophage I-J determinant biosynthesis after we destroyed these determinants

BioEssuys Vol. 4, No. 6 281


with t r y p ~ i n . ~ ~ Our data suggest that T lymphocytes but not macrophages syn- thesize thisdeterminant. The observation that macrophage membranes bind material shed by T lymphocytes would be consistent with the receptor hypothesis.

I 4 Determinants on Lipomodulin

AnI-J determinantwasrecentlyreported on an unusual lymph~kine.~ The lymphokine is secreted by T cells and macrophages; it inhibits the assembly of N-linked oligosaccharides to IgE-binding factors and exerts phospholipase- inhibitory activity. Monoclonal antibodies identified the lymphokine as a lipomodulin fragment. The authors suggest that the lipomodulin fragment may be an element of suppressive lymphokines.

An earlier report also described I-J determinants on lipomodulin.28 The combined reports suggest that testing suppressive lymphokines for phospholipase-inhibitory activity would be worthwhile. However, the data showing I-J determinants on macrophage-produced lipomodulin are inconsistent with the T-cell receptor hypothesis for I-J function.

Suppressor Cells may not Recognize H - 2 Gene Products

A final challenge to the receptor hypothesis is the observation that immune suppressor T cells may not recognize H-2 gene products. In this case their receptor proteins would not be expected to reflect H-2 polymorphisms. Several authors report that the suppressor T cell binds antigen alone. For some suppressor lymphokines, one cannot predict the strain distribution pattern of biological activity on the basis of H-2 genotype, as one can for other lymphokines. Thus the paradigm that immune T cells recognize foreign structures only in the context of self H-2 proteins may not be generalizable to suppressor T cells.

Conflicting Evidence from Chimera Experiments

A crucial test of the receptor hypothesis is to analyze the I-J phenotype of suppressor lymphokines derived from chimeric mice. Chimeras are produced by destroying the recipient's reticulo- endothelial system with radiation, then restoring it with undifferentiated lym- phoid stem cells of a different genotype.

If I-J epitopes reside on receptors for some H-2 gene product, then in chmeras the recipient's genotype, not the donor's, ought to govern the suppressive protein's I-J phenotype.

Four research groups have analyzed I-J determinants on proteins from chimeric mice (Table 111). Uracz and coworkers reported, with one exception, that I-J structures are controlled by the recipient's genotype.** Yamauchi and colleagues reported precisely the opposite.30 Sumida and others found a mixture of I-J determinant^,^^ and Noguchi et at. reported that the suppressor proteins had no I-J determinant at all.32 This crucial test of the receptor hypothesis will have to be repeated until a consensus is achieved and the inconsistencies explained.

Conclusion

A clear resolution for the I-J riddle has not yet emerged. Certain critical experiments would provide much-needed clues. Better biochemical characterization of molecules being termed I-J proteins would allow them to be compared. In particular, the biochemical nature of the determinant must be explored.

Testing assumptions made in the original genetic analysis would verify or refute them. In particular, further gene complementation experiments using the B104A(3R) strain in hybrid crosses would test the suggestion that this strain is not identical to C57BL/10 at loci outside H-2. Locating the Iu-4 structual gene by classic linkage tests would provide another important piece of the puzzle. DNA sequence data on the BIO.A(3R) and BIO.A(5R) strains in H-2 would support or refute the contention that they do not differ in H-2.

To support the receptor theory for I-J function, the conflict regarding I-J proteins on macrophages must be resolved. The chimera inconsistencies must beexplained. The paradigm regarding use of H-2-encoded elements by immune T cells must be extended to suppressor T cells, the relevant H-2 elements identified, and the nature of the suppressor T cell's receptor structure uncovered.

In my view, the resolution to the tangled I-J problem will probably involve an interplay among four H-2 loci, A@' A,, Es and E,, with the latter being pivotal. It will likely require at least one background locus expressed in macrophages, and at least twonon-allelic background loci expressed in suppressor T cells. Whether I-J is the hallmark of

282 BioEssays Vol. 4, No. 6


TABLE IIf. T lymphocytes from chimeras produce suppressor proteins whose I-J phenotypes are sometimes controlled by the recipient’s genome

Strain controlling the phenotype

Immunizing Functional Chimera antigen Assay system suppression I-J epitope Ref.

A+ B Hemocyanin

Hemocy anin +strain B antigen- presenting cells

Sheep red cells

None

Parent Sheep red +Fl cells

None

Fl+ Sheep red parent cells

None

IgG-secreting

IgG-secreting cells

cells

IgM-secreting

Allogeneic cells

mixed lymphocyte reaction

IgM-secreting cells

Allogeneic mixed lymphocyte reaction

IgM-secreting cells

Allogeneic mixed lymphocyte reaction

Donor

Donor and recipient (two distinct proteins)

Donor

n.d.

Recipient

n.d.

Recipient

n.d.

n.d.8

Donor and recipient (two distinct proteins)

None

Recipient

Donor

Donor (1G8 antibody)

Recipient (KN34 antibody)

Donor (single protein; both parental epitopes)

Recipient

31

31

32

29

30

29

30

29

n.d., not determined.

a suppressor T-cell receptor protein is still open to debate.

Solving the I-J problem will un- doubtedly uncover some fundamentals about suppressor T cells and immunoregulation. A decade’s work produced this intriguing if disquieting riddle. It will probably take more than a little time to unravel it.

I thank Dr Judith Kimble and members of my research group, Susan Smith, Martin Hochstrasser, Leslie Pond and Eric Denkers for puzzling over this riddle with me and offering comments on the manuscript. I am grateful to the Leukemia Society of America and the National Institute for Allergy and Infectious Diseases for their generous financial support.

REFERENCES 1 MARRACK, P. & KAPPLER, J. (1986). The T cell and its receptor. Sci. Amer. 254, 3-5. 2 TADA, T. & ASANO, Y. (1986). Somatic generation of a genetic polymorphism - towards the solution for the enigmatic I-J. BioEssays 4, 283-285. 3 MOLLER, G., ed. (1985). I-J. Immunol. Rev. 83. 4 MURPHY, D. B. (1978). The I-J subregion of the murine H-2 gene complex. Springer Seminars Immunopathol. 1, 11 1-131. 5 JARDIEU, P., AKASAKI, M. t ISHIZAKA, K.

(1986). Association of I-J determinants with lipomodulin/macrocortin. Proc. Natl. Acad. Sci. USA 83, 16&164. 6 HEALY, C. T., KAPP, J. A. &WEBB, D. R. (1983). Purification and biochemical analysis of antigen-specific suppressor factors ob- tained from the supernatant, membrane, or cytosol of a T cell hybridoma. J. Immunol.

7 KUMAGAI, Y., OKUMURA, K. & TADA, T. (1984). Photoaffinity-labeled hapten-binding T-cell receptor on a suppressor T-cell hybridoma. Molec. Immunol. 21, 545559. 8 TAUSSIG, M. J. & HOLLIMAN, A. (1979). Structure of an antigen-specific suppressor factor produced by a hybrid T-cell line. Nature 277, 308-3 10. 9 TANIGUCHI, M., SAITO, T., TAKEI, I. & TOKUHISA, T. (1981). Presence of interchain disulfide bonds between two gene products that compose the secreted form of an antigen-specific suppressor factor. J. Exp. Med. 153, 1672-1677. 10 SAITO, T. & TANIGUCHI, M. (1984). Chemical features of an antigen-specific suppressor T cell factor composed of two polypeptide chains. J. Mol. Cell. Immunol. 1,

11 GREENE, M. I., BACH, B. & BENACERRAF, B. (1979). Mechanisms of regulation of cell mediatedimmunity . 111. The characterization of azobenzenearsonate (ABA) specific suppressor T-cell-derived-suppressor factors (SF). .I. Exp. Med. 149, 1069-1083. 12 TADA, T., TANIGUCHI, M. & DAVID, C.S. (1976). Suppressive and enhancing T-cell factors as I-region gene products : properties

131,2843-2847.

137-145.

and the subregion assignment. Cold Spring Harbor Symp. Quant. Biol. 41, 119-127. 13 UPP, J. A., ARANEO, B. A. & CLEVIN- GER, B. L. (1980). Suppression of antibody and T cell proliferative responses to L-glutamic a~id~~-~-alanine3~-~-tyrosine~~ (GAT) by a specific monoclonal T cell factor. J. Exp. Med. 152,235-240. 14 KONTIAINEN, S., SIMPSON, E., BOHRER, E., BEVERLEY, P. C. L., HERZENBERG, L. A., FITZPATRICK, W. C., VOGT, P., TORANO, A., MCKENZIE, I. F. C. & FELDMANN, M. (1978). T cell lines producing antigen-specific suppressor factor. Nature 214,477430. 15 TURCK, C. W., KAPP, J. A. & WEBB, D. R. (1985). Purification and partial characterization of a monoclonal ‘ second order ’ suppressor factor specific for L-glutamic acids0-~-alanine30-~-tyrosine10. J. Immunol.

16 KLYCZEK, K. K., CANTOR, H. 8c HAYES, C. E. (1984). T cell surface I-J glycoprotein, concerted action of chromosome-4 and -17 genes forms an epitope dependent on a-D-mannosyl residues. J. Exp. Med. 159, 1604-1 6 1 7. 17 STIMPFLING, J.H. & RICHARDSON, A. (1965). Recombination within the histocompatibility 2 locus of the mouse. Genetics 51,

18 STEINMETZ, M., MINARD, K., HORVATH, S., MCNICHOLS, J., FRELINGER, J., WAKE, C., LONG, E., MACH, M. & HOOD, L. (1982). A molecular map of the immune response region from the major histocompatibility complex of the mouse. Nature 300,35-42. 19 KRONENBERG, M., STEINMETZ, M., KOBORI, J., KRAIG, E., KAPP, J. A., PIERCE, C. W., SUZUKI, G., TADA, T. & HOOD, L. (1983). RNA transcripts for I-J polypeptides are apparently not encoded between the I-A and I-E subregions of the murine major histocompatibility complex. Proc. Natl. Acad. Sci. USA 80, 5704-5708. 20 JAULIN, C., PERRIN, A., ABASTADO, J.-P., DUMAS, B., PAPAMATHEAKIS, J. & KOURILSKY, P. (1985). Polymorphism in mouse and human class I H-2 and HLA genes is not the result of random independent point muta- tions. Zmmunogenetics 22, 453470. 21 HAYES, C. E., KLYCZEK, K. K., KRUM, D. P., WHITCOMB, R. M., HULLETT, D. A. & CANTOR, H. (1984). Chromosome 4 Jr gene controls murine T cell surface I-J expression. Science 223, 559-563. 22 SCHWARTZ, R. H. (1985). T-lymphocyte recognition of antigen in association with gene productsofthemajor histocompatibility complex. Annu. Rev. Immunol. 3, 237-261. 23 SCHRADER, 3. W. (1979). Nature of the T-cell receptor. Scand. J. Immunol. 10,

24 HAYES, C. E. & KLYCZEK, K. K. (1985). The 1-1 glycoprotein: genetic control, biochemistry, and function. Immunol. Reu.

25 NIEDERHUBER, J. E. & ALLEN, P. (1980). Roleofl-region gene products in macrophage induction of an antibody response. 11. Restriction at the level of T cell in recognition of I-J-subregion macrophage determinants. J . Exp. Med. 151, 1103-1 113.

135,3232-3237.

831-846.

387-393.

83,41-59.

BioEssays Vol. 4, No. 6 283


26 MURPHY, D. B., YAMAUCHI, K., HABU, S., EARDLEY, D. D. & GERSHON, R. K. (1981). T cells in a suppressor circuit and non-T: non-B cells bear different I-J determinants. Immunogenetics 13, 205-213. 27 USUI, M., AOKI, I., SUNSHINE, G. H. & DORF, M. E. (1984). A role for macrophages in suppressor cell induction. J. Immunol.

28 HIRATA, F. & IWATA, M. (1983). Role of lipomodulin, a phospholipase inhibitory protein, in immunoregulation by thymocytes. J. Immunol. 130, 1930-1936. 29 URACZ, W., ASANO, Y., ABE, R. & TADA, T. (1985). I-J epitopes are adaptively acquired by T cells differentiated in the chimaenc condition. Nature 316, 741-743.

132, 1728-1734.

30 YAMAUCHI, K., FLOOD, P. M., SINGER, A. & GERSHON, R. K. (1983). Homologies between cell interaction molecules controlled by major histocompatibility complex- and Ig-V-linked genes that T cells use for communication; both molecules undergo 'adaptive' differentiation in the thymus. Eur. J. Immunol. 13,285-291. 31 SUMIDA, T., SADO, T., KOJIMA, M., ONO, K., KAMISAKU, H. & TANIGUCHI, M. (1 985). I-J as an idiotype of the recognition component of antigen-specific suppressor T-cell factor. Nature 316, 738-741. 32 NOGUCHI, M., ONOE, K., OGASAWARA, M., IWABUCHI, K., GENG, L., OGASAWARA, K., GOOD, R. A. & MOIUKAWA, K. (1985). H-2-incompatible bone marrow chimeras

produce donor-H-2-restricted Ly-2 suppressor T-cell factor(s). Proc. Natl. A c Q ~ . Sci.

33 STEINMETZ, M., STEPHAN, D. & FISCHER LINDAHL, K. (1986). Gene organization and recombinational hot spots in the murine major histocompatibility complex. CeN (in press).

USA 82,7063-7067.

COLLEEN E. HAYES is at the Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53106, USA.

Somatic Generation of a Genetic Polymorphism: Towards the Solution of the 14 Enigma Tomio Tada and Yoshihiro Asano

Sum ma ry

I-J has been regarded as a polymorphic genetic marker controlled by a locus in the major histocompatibility complex (MHC) which is expressed only on functional Tcells. However, this antigenic determinant has been found not to be directly encoded by the MHC gene per se but is somatically generated according to the MHC of the cellular environment during ontogeny. This explains the apparent linkage of I-J to MHC, despite the failure to find the structural gene for I-J within the MHC. The I-J paradox provides an instance of a polymorphism that does not follow the standard genetics of Mendelian structural genes.

Introduction

One of the greatest puzzles in contem- porary immunology is concerned with the I-J locus in the murine H-2 major histocompatibility complex (MHC). I-J was first defined as a locus in MHC that encodes a serologically detectable alloantigen on suppressor T cells (Ts) and soluble suppressor factors (TsF).'. Genetic polymorphisms for this locus associated with H-2 haplotypes have been firmly established by the use of monoclonal anti-I-J antibodies. Furthermore, production of a large number of I-J positive T-cell hybrid-

omas and clones with known functions enabled us to precipitate I-J polypeptide chains from the membrane lysate. These results led to the notion that I-J is a genetic marker encoded by a gene which maps in the immune response (Ir) region of murine MHC. This chromosomal region is known to encode polymorphic molecules called I a or class I1 antigens that arecomposed ofa andppolypeptide chains. The importance of class I1 antigens in the immune responses and immune cell interactions has been well established. The occurrence of two sets of recombinant mice, i.e. B10. A(3R) and B10.A(5R), and B10.HTT and B10. S(9R), allowed us to map the I-J locus as intercalated between previously known I-A and I-E subregions. I-J was, thus, about to gain citizenship as a unique member of I region gene products - a conclusion supported by hun- dreds of papers dealing with the functions and serology of the I-J-bearing molecules.

It was therefore a bolt from the blue when molecular analyses of DNA from the I region of MHC revealed that there exist no such genes coding for I-J alloantigen at the exactly prescribed po~it ion.~. When those recombinant mice differing in 1-J alloantigens were examined for their DNA polymorphism, no differences sufficient to explain the previously observed I-J allospecificities

were de t e~ ted .~ Subsequent detailed analyses clearly established that the I-J locus does not exist within MHC, even though the I-J products are still demon- strable with characteristic genetic polymorphisms exactly following the allelic form of the H-2 haplotype, i.e. all H-2k mice have I-J of k-type (I-Jk) and all H-2bmice I-Jb, and H-2k F, both I-Jk and I-Jb.

Exploration of the I 4 enigma

Immunologists today should feel lucky when they encounter such a first-rate paradox. Accordingly, there were inter- esting debates on the nature of I-J,6 inasmuch as no one was able to determine the exact structure of the molecule either by conventional biochemical means or by gene cloning techniques, probably reflecting the minute amounts of I-J molecules. Without referring to all these contro- versial debates, we want to discuss some of our own experimental results which revealed an unexpected plasticity of this genetic marker.

Since I-J had been defined by the specificity of alloantibodies, we re- examined the effect of monoclonal anti-I-J on T cell functions and found the following unusual effects: anti-I-J monoclonal antibodies were found to block both the syngeneic and allogeneic

problem and paradigms: i–j: a rogue's riddle

Documents