Proc. Natl. Acad. Sci. USAVol. 73, No. 7, pp. 2486-2490, July 1976Immunology

Role of major histocompatibility complex gene products indelayed-type hypersensitivity

[T cell (thymus-derived)/macrophlage/Ir gene/cell-mediated immunity/altered self]

J. F. A. P. MILLER, M. A. VADAS, ALISON WHITELAW, AND JENNIFER GAMBLEThe Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Victoria 3050, Australia

Communicated by Baruj Benacerraf, May 7, 1976

ABSTRACT Sensitized thymus-derived (I) lymphocytes cantransfer delayed-type hypersensitivity (DTH) to naive mice onlyif there is identity at the major histocompatibility complex(MHC). The MHC region responsible differs according to theantigen used for sensitization. For transfer of DTH to fowlgamma globulin identity at I-A is necessary; for dinitrofluoro-benzene, however, identity at either K, D, or I region is suffi-cient. T cells of one genotype, sensitized in a chimeric envi-ronment, transferred DTH to both parental strains even thoughthese were MIC incompatible. However,T cells from F1 hybridmice, sensitized not in the F1 but in one parental strain, trans-ferred DTH only to that parental strain, not to the other, incontrast to F1 T cells sensitized in the F1 which could transferDTH to both parental strains. Macrophages pulsed with antigenin vitro could be used to sensitize syngeneic or semi-allogeneicmice for the transfer of DIH. Transfer was, however, successfulonly in the strain syngeneic to that from which the macrophageswere derived. Evidence is also presented that genetically low-responder mice can be made to exhibit DTH provided they arepretreated with cyclophosphamide two days before sensitiza-tion.When considered in toto these results strongly argue in favor

of the notion that there are receptors on activated T cells whichrecognize antigenic determinants and MHC gene products. Theimplications of these findings are discussed in relation to therole of macrophages in antigen presentation and to the possibleparallel evolution of MHC gene products and of T cell receptorsfor antigen.

Many activities of thymus-derived (T) cells are regulated bygenes of the major histocompatibility complex (MHC). Thus,in the guinea pig, compatibility at theMHC was required formacrophage-associated antigen initiation of proliferation ofsensitized lymphocytes (1). In the mouse, cooperative interac-tions between helper T cells and antibody-forming precursor(B) cells or macrophages required identity at the I region of theMHC, both in vivo (2) and in vitro (3). Specific lysis of virus-infected or chemically modified target cells occurred effectivelyonly when the cytotoxic T lymphocytes and their targets wereMHC compatible, the genes required for this interactionmapping in the K or D, but not I, regions (4, 5). By contrast,transfer of delayed-type hypersensitivity (DTH) to proteinantigens by sensitized T cells was successful only in I-A com-patible recipients (6). The inability to transfer DTH in in-compatible recipients could not be attributed to rejection of theinjected cells, to their total recruitment into areas such as thespleen, nor to their engagement in a mixed lymphocyte reaction(6). Thus, for example, DTH was successfully transferred from

Abbreviations: T lymphocytes, thymus-derived lymphocytes; B cell,bone-marrow-derived lymphocyte; DNFB, 2,4-dinitro-1-fluoroben-zene; DTH, delayed-type hypersensitivity; FGG, fowl gamma globulin;125I-dUrd, 5-[12Iliodo-2'-deoxyuridine; L/R l25I-dUrd uptake, ratioof radioactivity of left ear to radioactivity of right ear; MHC, majorhistocompatibility complex; TBMC, tetraparental bone marrow chi-

mera.

sensitized F1 mice to naive mice of the parental strain that arecompetent to reject the F1 cells.The requirement for compatibility at some genes of the

MHC for DTH transfer may be taken to indicate that effectiveinteraction between sensitized lymphocytes and cells presentingantigen (probably macrophages) are governed by cell surfacestructures coded by MHC genes. The genetic constraints im-posed on this interaction may be explained in one of two ways.(1) It is possible that the antigen binding receptor on the T cellhas a combining site directed towards a structure on the ma-crophage that represents an antigen-associated modificationof the I region determinant or, alternatively, an I regionmodification of the processed antigen. This, represented dia-grammatically in model I of Fig. 1, is based on the model fa-vored by Doherty, Blanden, and Zinkernagel (4) to explain thegenetic constraints imposed by the K and D regions of theMHC for cytotoxic T lymphocytes. (2) An alternative possibilityenvisages the T cell receptor as a compound, with one partbeing the combining site directed towards the antigen and theother part consisting of a cell interaction molecule coded by theI region which is not a receptor for antigen. The T cell wouldbecome effective only if two reactions occurred at its surface:one would be the binding of the antigen to the combining site,and the other would be some conformational change inducedin the cell interaction molecule after matching another identicalmolecule on the surface of the macrophage. This model (shownas model II in Fig. 1) is based on the postulate by Katz and Be-nacerraf (2) of the existence of cell interaction molecules toexplain the failure of cooperative interactions between allo-geneic T and B cells in secondary antibody responses.We report here experiments, using three different ap-

proaches, that indicate that T cells involved in DTH perceiveboth MHC-coded cell surface molecules and processed anti-gen.

MATERIALS AND METHODSMice. Female mice (2- to 3-month-old) were used for sen-

sitization and as recipients of sensitized cells. Highly inbredspecific pathogen-free CBA/CaH Wehi, C57BL/6JWehi, andBALB/cAnBrWehi, and F1 hybrids between CBA and C57BLand between CBA and BALB/c mice were maintained inconventional rooms for the duration of the experiments. The.congenic lines of A.TH and A.TL were bred in conventionalrooms in our Institute. Male and female athymic CBA.nu andBALB/c.nu mice derived from the seventh to eighth backcrossgeneration of nude (nu/nu) mice to CBA or BALB/c wereused.T Cell Reconstitution of nu/nu Mice. The mice were given,

on three occasions 1 week apart, a mixture of syngeneic or (CBAX BALB/c)Fl thymus and lymph node cells intraperitoneally.During this interval the mice were no longer in the specific

2486

Proc. Natl. Acad. Sci. USA 73 (1976) 2487

Table 1. Identity at the D and K regions allows DTH transfer to DNFB but not to FGG

MHC of recipients* L/R '25I-dUrd uptaketSource of

sensitized cells Recipients K I-A I-B I-C S D DNFB FGG

A.TL A.TL s k k k k d 3.70 ± 0.79 1.96 ± 0.12A.TL A.TH s s s s s d 2.58 ± 0.30 1.20 ± 0.04A.SWt A.TL s k k k k d 2.03 ± 0.06 1.15 ± 0.07A.SW A.TH s s s s s d 2.78 ± 0.60 1.76 ± 0.16C57BL C57BL b b b b b b 5.50 ± 0.53 2.67 ± 0.26C57BL B1O.A(2R) k k k d d b 3.21 ± 0.34 1.12 ± 0.03CBA CBA k k k k k k 2.06 ± 0.10 2.48 ± 0.16CBA A.TL s k k k k d 2.72 ± 0.21 2.36 ± 0.28

* Letters in italic bold face point to region of difference between donor and recipient mice.t Values are arithmetic mean + 1 SEM. Five to six mice per group.t TheMHCofA.SWmiceisssssss.

pathogen free unit but were kept in sterile cages and givensterile food and water.

Tetraparental Bone Marrow Chimeras (TBMC). TBMCwere obtained by injecting lethally irradiated (950 roentgens,0.245 coulombs/kg) (CBA X C57BL)F1 hybrids with 107anti-Thy-l-treated bone marrow cells from both parentalstrains. These mice were kept, in specific pathogen free envi-ronment, for 2 months before use.

Sensitization. Two days before sensitization mice were given200 mg/kg (reduced to 150 mg/kg for nu/nu mice) of cyclo-phosphamide (Endoxan-Asta) subcutaneously to allow maximallevels of sensitization to all forms of antigen. Sensitization tofowl gamma globulin (FGG) and dinitrofluorobenzene (DNFB)have been described previously (6). For sensitization to the Bisoenzyme of porcine lactic dehydrogenase, the antigen wasemulsified in complete Freund's adjuvant and 0.1 ml (con-taining 20,ug of the isoenzyme) was injected into the two hindfootpads and subcutaneously into the abdominal region.

For sensitization with macrophage-associated antigen, 107mouse peritoneal exudate cells (75-85% macrophages) wereincubated at 370 for 60 min in 1 ml of Hanks' balanced saltsolution containing 10% fetal calf serum and 100lug of FGG.The cells were washed four times in cold medium and counted.The residual antigen has been estimated to be 0.1-0.3 ug/3 X106 macrophages. Viable antigen-pulsed macrophages (3 X 106)were then injected intravenously into cyclophosphamide pre-treated mice.

Transfer of Sensitized State. For transfer, peripheral lymph

1 ceN receptorfor gfta.ed

sew

MM00L ItCompoundT cell

* weptn

(Strain X) (stroin X)

-Ag CI * Acombining ind wcde combiningsite site

(Trui.X O rin

I(Strin)IvMI

-A^9FIG. 1. Hypothetical models used to explain genetic constraints

imposed on T lymphocyte-macrophage interactions (see text). X andY indicate hypothetical MHC- incompatible strains of mice. I, I-regiondeterminant "a" or "b"; MO, macrophage; Ag, antigen "1". A thirdmodel, mentioned in the text, is not shown here.

node cells and spleen cells were taken from mice 5 days aftersensitization and injected intravenously into naive mice (6).

Test for DTH. The sensitized mice and naive recipients ofsensitized cells were tested for DTH by the radioisotopic earmethod with use of 5-['25I]iodo-2'-deoxyuridine ('25I-dUrd)and radiometry as described previously (6). Briefly, the micewere challenged with antigen in the left ear and then injectedintraperitoneally with 0.1 ml of 1 mM 5-fluorodeoxyuridinefollowed by 2 1ACi of 125I-dUrd (specific activity 90-110 AiCi/g,Radiochemical Centre, Amersham, U.K.). The mice were killed24 hr later and the ears removed and counted in a PackardGamma Spectrometer. The results are expressed as the ratio ofthe radioactivity in the left ear to the radioactivity in the rightear (L/R '25I-dUrd uptake). It is unusual to obtain a ratiogreater than 1.2 in nonsensitized miceor in naive mice not re-ceiving sensitized cells.

Antisera. Anti-Thy-i and anti-H-2 sera of appropriatespecificities were prepared as described before (7).

RESULTSPrevious work establishing that the transfer of DTH requiredI-A identity at the MHC, at least for protein antigens such asFGG (6), is confirmed with mice used in an experiment re-ported in Table 1. In addition, whereas I-A identity alone wassufficient for DTH transfer to FGG, either I, K, or D identityalone could permit DTH transfer to DNFB (Table 1). Thisdifference in genetic constraints imposed on the transfer ofsensitivity to protein antigens and to reactive contact chemicalagents may be related to different subsets of T cells, some re-acting to antigens processed and presented by macrophages,and others reacting to compounds able to modify a variety ofMHC gene products on cell surfaces. The work establishing thedetails of these interactions will be described elsewhere.

DTH transfer with cells sensitized in chimericenvironmentsTo create an environment where a given antigen may be pre-sented on macrophages of two different genotypes, we usedtetraparental bone marrow chimeras (TBMC) of the type usedby von Boehmer and Sprent (8) in their studies on T and B cellcollaboration in antibody responses. Cells from such TBMC areunresponsive in both the mixed lymphocyte reaction and cellmediated lysis to either parent's histocompatibility antigens buthave unimpaired reactivity to third party determinants (8). Thecells of the TBMC do, however, express K and D serologicalspecificities of the MHC since they can be lysed by specificalloantisera, and also express I determinants since they can be

Immunology: Miller et al.

Proc. Nati. Acad. Sci. USA 73 (1976)

Table 2. T cells sensitized in chimeric environment transfer DTH to MIC-incompatible parental recipients

L/R 125I-dUrdSource of sensitized cells* Recipients uptaket

Cells from sensitized CBA - C57BL TBMC CBAt 1.45 ± 0.03treated with anti-C57BL H-2 serum (CBA x C57BL)F, 1.82 ± 0.12

C57BL 1.91 ± 0.10

CBA cells from sensitized CBA CBA 1.98 ± 0.06(CBA x C57BL)F, 1.89 ± 0.05

C57BL 1.01 ± 0.05

* Lymphoid cells (10 x 106) were transferred.t The response to FGG in recipients not given cells was CBA: 1.10 i 0.04; (CBA x C57BL)F1: 1.12 + 0.07; C57BL: 1.13 + 0.02.t Arithmetic mean X 1 SEM. Five mice per group.

used as stimulator cells in the mixed lymphocyte reaction (8).TBMC made from CBA and C57BL mice and containingroughly 50% CBA cells and 50% C57BL cells in their hemo-poietic system, were sensitized to FGG. Lymph node cells weretaken 5 days later, and separated into CBA and C57BL cells byappropriate incubation with relevant anti-H-2 sera able to killeither CBA or C57BL cells. The remaining cells were thenwashed and tested for their capacity to transfer DTH to naiverecipients. For simplicity, we show here the results obtainedwith the transfer of CBA cells sensitized to FGG in the chimericenvironment (Table 2). The cells transferred DTH as effectivelyto allogeneic C57BL as to syngeneic or F1 mice. This contrastswith the failure of CBA cells, sensitized in normal nonchimericCBA mice, to transfer DTH to allogeneic C57BL mice. Thesefindings argue in favor of the notion that T cells, primed in a,chimeric environment made up of two different strains, are

primed to antigen 1 and I region gene product a (Fig. 2) as wellas to antigen 1 and I region gene product b. Thus, in the TBMCenvironment, as suggested by von Boeheer and Sprent (8), CBAT cells would be composed of two different subsets of primedT cells, one with specificity towards the antigen and the I regionof CBA and another with specificity towards the antigen andthe I region determinant of C57BL. Alternatively, the T cellscould utilize two receptors, one recognizing antigen, the otheran I-region gene product.DTH transfer with F1 cells sensitized in parental strain

It was previously shown (6) that primed T cells from F1 micewill successfully transfer DTH to both parental strains as wellas to the syngeneic F1. The question was therefore asked: can

F1 T cells sensitized, not in the F1 animal, but in one of theparental strains (say X) transfer DTH to both parental strains(X and say Y) or only to that in which they were originallysensitized, namely X. To sensitize F1 cells not in the F1 but inone of the parental strains, we made use of the athymic nudemice which are available on several congenic backgrounds.DTH could not be elicited in these mice unless they were first

*- TBMC X*OY--

(stram X) I(strainY)IM M

-- jr'i00 -0 A

Ag-. -Agcombining sombining

T cell T cell(stroln X) (strain X)

2 subsets of primed X T cells& 2 subsets of primed Y T cells

FIG. 2. Possible different types of reactive T cells in sensitizedtetraparental bone marrow chimeras (TBMC) (see text). Abbrevia-tions as in Fig. 1.

reconstituted with thymus cells or a mixture of thymus andlymph node cells (Table 3). To sensitize F1 T cells in the pa-rental environment, T cells from naive, nonsensitized, (CBAX BALB/c)F1 mice were used to reconstitute BALB/c.nu andCBA.nu, and these were then sensitized to FGG. We thentransferred lymph node cells from such reconstituted mice tonaive BALB/c and CBA mice to test for their ability to transferDTH. It is evident that F1 cells sensitized in BALB/c.nu couldtransfer DTH successfully only to BALB/c, not to CBA. On theother hand, F1 T cells sensitized in CBA.nu could transfer DTHsuccessfully only to CBA, not to BALB/c (Table 4). These resultsstrongly support the notion that sensitization of T cells in an F1mouse leads to the activation of two subsets of F1 T cells withrespect to the specificity of their antigen combining sites: onewith sites directed towards the antigen and the I-A. region geneproduct of one parental strain, the.other with sites directedtowards the antigen and the I-A region gene product of theother parental strain (Fig. 3). Sensitization of F1 T cells in theparental environment would thus lead to the activation of onlyone of these two subsets of T cells. The model (model II, Fig.1) which demands matching of identical I-A region geneproducts can be substantiated only if these products weresubject to allelic exclusion, for which there is no evidence.

DTH transfer with cells from mice sensitized byantigen-pulsed macrophagesMacrophages from CBA or BALB/c mice were pulsed withFGG in vitro, washed and injected into cyclophosphamide-pretreated (CBA X BALB/c)F1 mice. High levels of sensitivitywere achieved in this manner with doses of antigen that werecalculated (using radiolabeled FGG) to be 100 to 1000 less thanis needed for aggregated antigen. Lymphoid cells from micesensitized with such macrophage preparations were tested fortheir ability to transfer DTH into parental naive recipients. Theresults shown in Table 5 indicate that cell transfer of DTH waspossible provided the macrophages used for sensitization were

Table 3. DTH to FGG in athymic- nude mice

Strain of L/R '2SI-dUrdnude mice T cells given* uptaket'BALB/c.nu None 1.05 ± 0.11BALB/c.nu BALB/c 4.10 ± 0.06BALB/c.nu (CBA x BALB/c)F, 3.82 ± 0.16CBA.nu None 0.94 ± 0.08CBA.nu CBA 3.23 ± 0.50CBA.nu (CBA x BALB/c)F, 3.70 ± 0.30

* Mice were given a mixture of 4 X 108 thymus and lymph nodecells in two doses 1 week apart.

t Arithmetic mean 4 1 SEM. Five to six mice per group.

2488 Immunology: Miller et al.

Proc. Natl. Acad. Sci. USA 73 (1976) 2489

Table 4. Effect of transfer of sensitized lymph node cellsfrom reconstituted nude mice

Donors of sensitized L/R '25I-dUrdlymph node cells* Recipients uptaket

CBA.nu CBA 1.65 ± 0.08 (5)CBA.nu BALB/c 1.18 ± 0.13 (5)BALB/c.nu CBA 1.01 ± 0.15 (4)BALB/c.nu BALB/c 1.73 ± 0.14 (4)

CBA 1.17 ± 0.09 (6)BALB/c 1.15 ± 0.04'(6)

* Donors were reconstituted by 3 weekly injections of 108 (CBA xBALB/c)Fi thymus + mesenteric lymph node cells before sensi-tization. Lymph node cells (17 x 106) were injected intravenouslyinto each recipient.

t Mean + SlM. Number of mice per group shown in brackets.

of the same genotype as the recipients. This again stronglysupports the notion that sensitization is directed not to the an-tigen as such but to cell surface structures on the macrophagedetermined partly by anMHC gene product (I-A) and partlyby the processed antigen. Similar observations have recentlybeen made by Pierce et al. (9) who demonstrated restrictionin macrophage-T cell interaction in the secondary responseinduced by the primary response.DTH in responder and nonresponder miceThe above results suggest that one possible mechanism of Irgene control of T cell-dependent responses is that low re-sponders may lack the appropriate I region gene that is essentialto produce (in the macrophage) the product with which aparticular antigenic determinant can become associated afterthe antigen is processed by that cell. The defect in the low re-sponder may thus depend on an inadequate repertoire of I-Aregion gene products expressed in macrophages. Alternatively,the defect could be in the scope of the receptor dictionary inthe T cells. It was, therefore, of interest to study the respon-siveness of mice to the B isoenzyme of lactic dehydrogenasewhich is under genetic control (10). As shown in Fig. 4, a goodDTH response was obtained in responder C57BL mice butnone, at any time, in nonresponder CBA mice. If, however, themice were pretreated 2 days prior to antigen sensitization with200 mg/kg of cyclophosphamide, DTH could be elicited fromCBA mice, but the period of sensitivity was short lived. The lowresponder strain cannot therefore lack T cells with appropriatespecificities, nor the mechanism by which stimulator cells ormacrophages effectively present antigen to T cells (presumablyin association with the I-A region gene product). Under normalconditions however, this stimulation potential is suppressed by

I (X/Yb)Fi I

a l A 9

T oell T cell(anti-cl) (anti-bl)

(Xa/Yb) Fi (XaYbF2 sub se ts of anti-I reactive T cells

In primed FhFIG. 3. Possible different types of reactive T cells in sensitized

F1 (see text). Abbreviations as in Fig. 1.

a cyclophosphamide-sensitive mechanism, the nature of whichremains to be elucidated. Candidates for such a suppressivemechanism are, of course, suppressor T cells but their role inthis particular system has yet to be established unequivocal-ly.

DISCUSSIONTaken in toto, the findings obtained in the experiments withTBMC, with F1 T cells sensitized in parental strains, and withcells sensitized by semi-allogeneic macrophages, argue stronglyin favor of the notion that sensitized T cells have antigen re-

ceptors predominantly directed to cell surface components.These are determined partly by genes coding for the K, I, orD products and partly by the antigenic determinant as it ap-pears on the cell surface after macrophage processing. It wouldappear from our previous work with DTH to FGG (6) and fromthe work of Erb and Feldmann with helper T cells in antibodyproduction (3), that the Ly-1 T cell, which is responsible for bothDTH and helper functions (11), can be activated only by anti-gen associated with macrophages and can discriminate variationin antigenic pattern associated with I region gene products. Onthe other hand, the Ly-2 T cell, responsible for direct cytotox-icity, has a dictionary restricted to variations of the pattern ofantigen in association with molecules coded by theK orD genesof the MHC (4). From the data obtained here (Table 1), it istherefore tempting to postulate that two subsets of T cells maybe responsible for the transfer of DTH to DNFB.A basic difference in the mechanisms of antigen activation

of primed B and T cells was suggested by previous work per-formed here with the collaboration of Prof. Basten (7) in whichentirely different conditions were shown to be essential forinactivation or "suicide" of primed T and B lymphocytes byradiolabeled antigen. First, T cell suicide, unlike B cell suicide,could be achieved only if antigen was presented, not in solubleform, but in association with other cell types, probably mac-rophages. Second, suicide occurred with T cells only if incu-

Table 5. Transfer of DTH with lymphoid cells from mice sensitized with macrophage associated antigen

Naive recipient of6 X 107 sensitized

Source of FGG Mice sensitized with (CBA X BALB/c)F1pulsed macrophages FGG pulsed macrophages* spleen cells L/R 125I-dUrd uptaket

CBA (CBA x BALB/c)F, CBA 1.62 ±.0.09CBA (CBA X BALB/c)F, BALB/c 1.11 ± 012BALB/c (CBA X BALB/c)F1 CBA 1.12 ± 0.05BALB/c (CBA X BALB/c)F1 BALB/c 1.63 ± 0.17

* The L/R 1251-dUrd uptake in (CBA x BALB/c)F1 when sensitized with CBA macrophage-associated FGG was 3.71 4 0.29 while withBALB/c-associated FGG, it was 5.91 0.61 on the day of cell transfer.

t Arithmetic mean + 1 SEM. Six mice per group.

Immunology: Miller et al.

Proc. Natl. Acad. Sci. USA 73 (1976)

: 2-0

-

-J

L-I

lo0 .' Po a I a I a I I a 1- i I a I

J 3 S 7 9 it 13 Is 17 19,2 DAYS AFTER SENSITIZATION TO LDH B

FIG. 4. DTH response in C57BL and CBA mice sensitized withthe B isoenzyme of lactic dehydrogenase (LDHB). 0-0, response

in CBA mice not pretreated with cyclophosphamide; A-A, response

in CBA mice pretreated with cyclophosphamide; *-*, response inC57BL mice not pretreated with cyclophosphamide; A-A, response

in C57BL mice pretreated with cyclophosphamide. Controls were notsensitized.

bation was at 370, not at 4 ° even though 40 was effective forB cell suicide. Third, T cells could be protected from suicideby preincubation with alloantiserum directed against relevantMHC components whereas anti-immunoglobulin had no pro-

tective effect. By contrast, B cell suicide could be prevented byanti-immunoglobulin, not by anti-H-2 pretreatment. Finally,certain inhibitors of metabolism, such as azide, had no effecton B cell suicide but prevented T cell suicide. These findingssupport the notion that components of the MHC must somehowbe intimately involved in primed T cell activation. In addition,these observations may explain why so-called "T-independent"antigens (12) or antigens composed of D-amino acids (13) donot induce T helper cells or allow DTH, although they can elicitIgM responses from B cells. Such antigens are either poorlymetabolized or nondegradable and hence may be unable toactivate T cells since this appears to require antigen processingby metabolically active macrophages.

Since macrophages are essential for the activation of someT cell subsets, it is possible that the lesion of genetic nonres-

ponsiveness resides in the macrophage which could display aninadequate repertoire of I-A region gene products. This ideadoes not appear to be supported by the results obtained withthe B isoenzyme of lactic dehydrogenase in which low re-

sponders could be made to respond after cyclophosphamidetreatment. Alternatively, cyclophosphamide may temporarilyallow macrophages to process antigen in a more immunogenicform, irrespective of the responder status.

There is increasing evidence that evolution of cell surfacecomponents occurred so that recognition of nonself in theprocess of fertilization in hermaphrodite species and of self inthe process of cell association for the growth of particular tissueswas possible. The basis for such diversity of cell surface struc-tures in the colonial tunicate, Botryllus, is a single genetic locuswith multiple alleles (14). Such a gene locus is the logical pre-

cursor of the 4 loci on chromosome 17 of the mouse, Tit, H-2K,H-2D, and TL, each of which specifies surface glycoproteins*of similar nature and molecular weight and enables cell rec-ognition either in embryogenesis or later (15). If MHC geneproducts did evolve to enable cell recognition for allofertiliza-tion and for tissue growth and development, there must havebeen an associated parallel evolution of complementary rec-ognition elements. It is tempting to suggest that from these el-ements evolved the T cell receptors for antigen. The possibilitythat at least part of this receptor is coded by V genes similar oridentical to those responsible for immunoglobulin diversity hasreceived support from the recent work of Eichmann and Ra-jewsky (16) and Binz and Wigzell (17). This suggests, as an al-ternative to the "altered self" hypothesis (model I, Fig. 1), thatT cells may utilize a dual recognition system, i.e., two separatereceptors, one for antigen, the other for a particularMHC giveproduct (18). The results reported here do not discriminatebetween these two hypothesis, but they do exclude the mech-anism illustrated by model II of Fig. 1.

This work was supported by the National Health and Medical Re-search Council of Australia and by U.S. Public Health Service ResearchGrant no. 63992 from the National Cancer Institute.

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Shen, F.-W., Boyse, E. A., Gamble, J. R. & Whitelaw, A. M.(1976) J. Exp. Med. 144, in press.

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14. Burnet, F. M. (1971) Nature 232,230-235.15. Vitteta, E. S., Artzt, K., Bennett, D., Boyse, E. A. & Jacob, F.

(1975) Proc. Natl. Acad. Sd. USA 72,3215-3219.16. Eichmann, K. & Rajewsky, K. (1975) Eur. J. Immunol. 5,

661-666.17. Binz, H. & Wigzell, H. (1975) J. Exp. Med. 142, 1218-1230.18. Doherty, P. C., Gotze, D., Trinchieri, G. & Zinkernagel, R. M.

(1976) Immunogenetics, in press.

2490 Immunology: Miller et al.

r-