Proc. Nati Acad. Sci. USAVol. 78, No. 1, pp. 514-518, January 1981Immunology

Inhibition of antigen-induced proliferation ofT cells from radiation-induced bone marrow chimeras by a monoclonal antibody directedagainst an Ia determinant on the antigen-presenting cell

(Ia antigens/lr genes/T-cell repertoire)

DAN L. LONGO AND RONALD H. SCHWARTZLaboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205

Communicated by Richard M. Krause, September 18, 1980

ABSTRACT Chimeric BIO.A T cells that had matured in a(BlO.A x B10.Q)F1 environment acquired the ability to respond topoly(Glu'Lys35Phe9) (GL4), an antigen to which the BlO.A mouseis a nonresponder. The response of the chimeric BlO.A T cells wasinitiated by.GL4 on responder BlO.Q antigen-presenting cells(APC) but not by GL4W on nonresponder BlO.A APC. Similarly,chimeric BlO.Q T cells that had matured in a (BlO.A x BlO.Q)F,environment acquired the ability to respond to poly(Glu'Ala'Tyr'0)(GAT) when the antigen was presented on responder BIO.A APC,but not when GAT was presented on nonresponder BlO.Q APC.No syngeneic haplotype preference was observed for either anti-gen. These interactions between H-2 nonidentical T cells and APCwere inhibited by anti-H-2 antisera and a monoclonal anti-Ia an-tibody directed against the APC but not by such antibodies whenthey were directed against the T cell. These data suggest that,when they develop in a responder chimeric environment, geno-typic nonresponder T cells become responders by acquiring recep-tors that allow them to recognize responder I region products onthe surface of APC. Furthermore, the data demonstrate that thesite of action of the blocking effects of the anti-Ia antibodies is theAPC, thus providing strong evidence in support of the idea that Iaantigens on APC are the Ir gene products.

One approach to understanding the mechanism of immune re-sponse (Ir) gene function has been to determine the cell(s) inwhich the genes are expressed. Experiments with T cells fromF1 (responder x nonresponder) guinea pigs (1) and mice (2), ingeneral, have shown that Ir gene-controlled responses can beinitiated by antigen-pulsed responder antigen-presenting cells(APC) but not by antigen-pulsedsnonresponder APC. Thesefindings suggested that one cell type that must express re-sponder Ir gene products is the APC. Furthermore, the inter-action between F1 T cell and responder APC could be inhibitedby anti-Ia antisera specific for the Ia molecules encoded in thesame I subregion in which the Ir gene for the antigen mapped(3,4).

Experiments with radiation-induced bone marrow chimerasin general have confirmed the idea that the APC must be of re-sponder genotype to generate Ir gene-controlled responses(5-8). In addition, such chimeras have allowed the analysis ofthe requirements for Ir gene expression at the T-cell level in theabsence of allogeneic effects. Chimeric nonresponder T cellswhich have matured in a responder environment become phen-otypic responders, suggesting that the nonresponder T cell isnot defective simply because it does not possess the responderJr gene. In-an effort to learn more about the nature ofthe phen-otypic alteration ofnonresponder T cells in the responder envi-ronment, we made chimeras of the type B10.A + B10.Q -(B10.A x BIO.Q)Fl. B10.A mice are responders to poly-

(Glu6OAlaTyrl0) (GAT) and nonresponders to poly(Glu'Lys35Phe9) (GLO). The B1O.Q mice are responders to GLOW and non-responders to GAT. B10.A + B1O.Q -- (BIO.A X BLO.Q)F1chimeras were primed with GLUT and GAT, and the repertoireof each donor type T-cell population was assayed by killing theother donor type cells with anti-H-2 antisera and complementand adding back to the culture irradiated APC of the killed do-nor type. Such studies revealed that the chimeric nonresponderT cell becomes a phenotypic responder by learning to recognizeresponder major histocompatibility complex (MHC) productson the APC. This interaction could be inhibited by anti-Ia anti-sera specific for the APC, including a monoclonal antibody, butnot by anti-Ia antisera specific for the T cell. These experimentsprovide another argument in support of the idea that the Ia an-tigens on APC are products of the Ir gene.

MATERIALS AND METHODSAnimals. B1O.Q and (B10.A X B10.Q)F1 animals were bred

in our own animal colony from stocks originally supplied by J.H. Stimpfling (McLaughlin Research Institute, Great Falls,MT). B10.A mice were purchased from The Jackson Laboratory(Bar Harbor, ME). B1O.T(6R) mice were bred from stocks orig-inally supplied by David Sachs (Transplantation Biology Sec-tion, Immunology Branch, National Cancer Institute, Be-thesda, MD).

Chimeras. Radiation-induced bone marrow chimeras weremade as described (7). Briefly, (B1O.A X B10.Q)F1 mice weregiven 925-975 R (0.24-25 C/kg) from a heavily filtered x-raysource and reconstituted on the same day with 107 T cell-de-pleted bone marrow cells from one or both parents. T cell-de-pleted bone marrow was obtained by treating donor mice withanti-thymocyte antiserum and cortisone in vivo and rabbit anti-mouse brain plus guinea pig complement in vitro. The chimeraswere used no sooner than 3 months after irradiation. Spleencells from individual chimeras were H-2 typed before use andall were found to be entirely ofdonor origin. All B10.A + B10.Q-* (B10.A X B1O.Q)Fl chimeras were found to be balanced mix-tures ofboth donors (33-67%).

Antigens and Immunization. GL46 (originally purchasedfrom Miles-Yeda, Rehovot, Israel) was the generous gift ofAlanRosenthal (Merck, Rahway, NJ). GAT (lot 6) was purchasedfrom Miles-Yeda. Both antigens were emulsified in completeFreund's adjuvant containing 1 mg of Mycobacterium tubercu-losis strain H37Ra (Difco) per ml and administered to mice in

Abbreviations: GL4b, poly(Glu56Lys35Phe9); GAT, poly(Glu"OAWaTyr'l);APC, antigen-presenting cells; Ir gene, immune response gene; PPD,purified-protein derivative ofMycobacterium tuberculosis; MHC, ma-jor histocompatibility complex.

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Proc. Natl. Acad. Sci. USA 78 (1981) 515

both hind footpads and the base of the tail at a total dose of 30,ug per mouse. Both antigens were used in culture at a final con-centration of 100 ug/ml. Purified-protein derivative of Myco-bacterium tuberculosis (PPD) (Connaught Medical ResearchLaboratory, Willowdale, ON) was used in culture at 20 ,ug/ml.When single parent-* FI chimeras were immunized, 108 T cell-depleted F. bone marrow and spleen cells were given intrave-nously as a source of the other parental APC. These cells werekilled with appropriate antisera and complement before assay.

Antisera. (A/J x BI0.A)F1 anti-B10.Q (anti-Q) and (A.TH x

B1O.Q)Fl anti-B10.A (anti-A) antisera were made by repeatedintraperitoneal injection ofthe F1 mice with 107 B10.Q or B10.Aspleen cells (9). The anti-Q antiserum, which has specificity forthe entire H-2 region ofthe q haplotype, had a plateau cytotoxictiter for B10.Q lymph node and spleen cells of 1:512. The anti-A antiserum, which has specificity for the K, I, and S (but notD) regions of the a haplotype, had a plateau cytotoxic titer forB10.A lymph node and spleen cells of 1:512. Both antisera spe-

cifically inhibited T-cell proliferative responses of the strainagainst which they were reactive at a concentration in culture of1% (vol/vol). The anti-Q antiserum had no effect on B10.A Tcellproliferation at 1% concentration, and the anti-A antiserum hadno effect on B10.Q T-cell proliferation at 1% concentration.A.TH anti-A.TL (no.2081 from David Sachs), which has speci-ficity for Ik and Sk, inhibited B10.A T-cell proliferation at a con-centration of1% and had no effect on B10.QT-cell proliferation,presumably reflecting an absence of significant reactivityagainst Ia.3, a determinant shared by B10.A and B1O.Q. Thisresult was also confirmed by cytotoxicity testing on B10.Qspleen cells. A monoclonal antibody with specificity for Ia. 17was produced from cells of the hybridoma designated 10-2.16(10) which had been passaged in vitro in modified Eagle's me-

dium supplemented with 10% fetal calf serum after being ob-tained from the Salk Institute (La Jolla, CA). The cell culturesupernatant possessing a plateau cytotoxic titer of 1:256 againstB10.A spleen cells was the generous gift of Richard Hodes (Im-munology Branch, National Cancer Institute).

T-Cell Proliferation and Blocking Studies. B10.A + B10.Q(B10.A x B10.Q)F, chimeras that had been immunized 8

days previously were sacrificed by cervical dislocation and theirpopliteal, inguinal, and para-aortic lymph nodes were har-vested. Lymph node T cells eluted from nylon wool columns(11) were treated with either anti-A or anti-Q antiserum plus

rabbit complement in a two-stage cytotoxicity procedure. Thosecells remaining after treatment with anti-Q plus complementwere designated "chimeric B1O.A" cells and those remainingafter treatment with anti-A plus complement were designated"chimeric B1O.Q" cells. Chimeric lymph node cells (4 x 105)were plated in 96-well flat-bottomed microtiter plates alongwith soluble antigen and 108 irradiated (2000 R) spleen cellsfrom either parent. Each culture was pulsed with 1 ,Ci (1 Ci= 3.7 X 1O0 becquerels) of[3H]thymidine (6.7 Ci/mmol; NewEngland Nuclear) on day 4, and incorporation was measured18 hr later. The cpm data are expressed as arithmetic mean

±SEM.Inhibition studies were performed by adding appropriate

antisera to each culture at a final concentration of 1% (vol/vol).The antiserum was present throughout the culture period. Themonoclonal anti-Ia. 17 antibody was used at a final concentrationof 10%.

RESULTS

The responsiveness of B1O.A + B1O.Q -- (B1O.A X B1O.Q)F1chimeric T cells to GAT and GL4 was assayed. Not unexpect-edly, these T cells, which were a balanced mixture ofB10.A andB1O.Q cells, responded well to both antigens as well as to PPD(Table 1). The responses were augmented by the addition of ir-radiated spleen cells from both donors, presumably reflectingthat APC are limiting under the conditions of our lymph nodeT-cell isolation and assay.To examine each donor haplotype T cell separately, chimeric

lymph node T cells were treated with anti-Q antiserum pluscomplement to isolate chimeric B1O.A T cells and with anti-Aantiserum plus complement to isolate chimeric B1O.Q T cells.Chimeric B1O.A T cells, which after the antiserum treatmentare in the presence of only B1O.A APC, responded to GAT butnot to GL4 (Table 1). However, when GL4-responder APCwere added in the form of irradiated BLO.Q spleen cells, a sub-stantial GL4 response appeared. In addition, the PPD re-

sponse was augmented by adding B1O.Q spleen cells, suggest-ing that a portion ofthe total proliferation ofthe chimeric B10.AT cells to this antigen, the response to which is not under Irgene control, is specific for PPD in association with the H-2-dif-ferent B1O.Q restriction elements. The addition of B1O.Q APCfailed to augment the response of the chimeric B10.A T cells to

Table 1. Expansion ofthe repertoire ofchimeric T cells to interact across an H-2 barrier and the inhibition ofsuchinteractions with antisera directed against the APC, not the T cell

Cells Medium PPD GL4 GAT

A + Q - (A x Q)Fj T cells 1127 ± 184 23,691 ± 1,348 29,677 ± 2,282 34,922 + 2,742+ added A and Q spleen 1918 ± 201 66,319 ± 6,103 75,811 + 6,578 82,462 ± 7,055

ChimericATcells 925 ± 146 20,754 ± 1,961 887 ± 116 63,381 ± 4,123Chimeric AT cells + Q spleen 1643 ± 127 61,726 ± 5,513 69,959 + 6,329 56,229 ± 3,038+ anti-Q antiserum 1007 ± 84 24,326 ± 926 2,937 ± 129 61,665 ± 2,946+ anti-A antiserum 827 ± 163 29,378 ± 1,657 72,216 ± 5,308 2,188 ± 144

Chimeric Q T cells 1093 ± 123 27,233 ± 1,468 53,761 ± 4,117 1,966 ± 214Chimeric Q T cells + A spleen 2109 ± 191 68,459 ± 5,207 48,477 ± 3,048 89,217 ± 5,002+ anti-A antiserum 911 ± 88 31,267 ± 3,326 50,925 ± 1,866 2,724 ± 251+ anti-Q antiserum 1234 ± 92 26,958 + 2,042 2,254 + 118 78,041 ± 4,421

B1O.A + B1O.Q -. (B1O.A x B1O.Q)F1 chimeras were immunized and their lymph nodes were harvested 8 days later. Afterpassage over nylon wool columns, lymph node T cells were plated directly or with added excess B1O.A and B1O.Q spleen cells asa source ofAPC. Chimeric B1O.A T cells were obtained by treating the chimeric cells with anti-Q antiserum plus complementand these cells were either plated directly or with added B1O.Q spleen. The interaction between the chimeric B1O.A T cells andB1O.Q APC was then probed by using blocking antisera directed against the T cell or the APC. Chimeric B1O.Q T cells wereisolated by treating chimeric T cells with anti-A plus complement and the cells were plated directly or with added B1O.A spleencells. Attempts to block their interaction with anti-A antiserum or anti-Q antiserum were performed. Each antigen was addedat 100 pg/ml and the antisera for blocking were added at a final concentration of 1%.

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516 Immunology: Longo and Schwartz

GAT, an antigen to which the B1O.Q is a nonresponder. These

results demonstrate that the chimeric B10.A T cells are capableof recognizing GL4 in association with B10.Q but not B1O.AAPC, GAT in association with B1O.A but not BLO.Q APC, andPPD in association with both B1O.A and B10.Q APC. The datasuggest that B10.A T cells, which are genotypic GL4 nonre-

sponders, become GL4 responders when they mature in a re-

sponder (B1O.Q) environment by learning to recognize re-

sponder MHC products as self.To clarify the nature of the interaction between chimeric

B10.A T cells and B1O.Q APC, blocking antisera were added tothe cultures. The addition of an anti-Q antiserum directed onlyagainst the added B1O.Q APC virtually eliminated the GL4 re-

sponse of the chimeric BIO.A T cells (Table 1). The same anti-serum partially inhibited the PPD response, which could be in-itiated by either B1O.A or B1O.Q APC, and had no significanteffect on the GAT response, which was initiated only by re-

sponder B10.A APC. In contrast, anti-A antiserum, which was

capable of reacting against the responding chimeric B10.A Tcells, failed to inhibit the GL4E response. The anti-A antiserumwas functional because it partially inhibited the response toPPD and almost completely eliminated the response to GAT.Because the anti-A antiserum failed to inhibit the GL4E responseof the B1O.A T cells which could be initiated only by B10.QAPC, the partial block of the PPD response and the completeinhibition ofthe GAT response must represent the effects oftheantiserum on the B1O.A APC, which participate in these lattertwo responses, and not effects on the B10.A T cells. Overall,these data demonstrate that responsiveness cannot be inhibitedby anti-MHC antisera directed against the proliferating T cellbut can be virtually eliminated by antisera specific for the re-

sponder APC. Antisera specific for a nonresponder APC haveno effect because these cells do not participate in the immuneresponse. Thus, the inhibition data support the concept thatgenotypic nonresponder chimericT cells become responders byacquiring the capacity to recognize responder MHC productson APC.The complete reciprocal experiment is shown in the last four

lines of Table 1. Treatment with anti-A antiserum and comple-ment to remove all B10.A T cells and APC left chimeric B10.QT cells and APC which could respond to GL4 and PPD but notto GAT, an antigen to which the BlO.Q is a low responder. How-ever, when GAT-responder B10.A APC were added, a largeGAT response appeared and the PPD response approximatelydoubled in magnitude. These results demonstrate that the chi-meric B10.Q T cells can respond to GAT but only in associationwith B10.A APC, whereas the T cells can respond to PPD in as-

sociation with both B1O.A and B10.Q APC. The GAT responseof the chimeric B1O.Q T cells was inhibited by anti-A antiserumspecific for the responder B10.A APC but not by anti-Q anti-serum directed against the T cell and nonresponder APC. Theanti-Q antiserum did inhibit almost completely the GL4 re-

sponse which requires B1O.Q APC. Once again it was observedthat a portion of the T cell response to PPD was inhibited byboth anti-A and anti-Q antisera, suggesting that for this antigen,to which both B1O.A and B1O.Q are responders, the chimericB10.Q T cell is as likely to interact with B10.A as with B10.QAPC. No haplotype preference for the genotypically identicalMHC products (H-2q) on APC was seen.

Previous blocking experiments with both guinea pig (12) andmouse (3) T-cell proliferative responses have suggested that theantiserum inhibits the T cell-APC interaction through its anti-Ia specificities. To determine whether the same was true for thechimeric cells, the experiment in Table 1 was repeated with an

A.TH anti-A.TL antiserum which is specific for Ik and Sk deter-minants. Because the BLO.A strain possesses d alleles to the

Table 2. Anti-Ia antisera have effects on chimeric T cell-APCinteractions similar to those of anti-H-2 antisera

Cells Medium GAT GLOChimeric QT cells 2250 ± 194 5,084 ± 429 15,391 ± 3,221

Chimeric QT cells +A spleen 3478 ± 93 31,710 ± 870 17,035 ± 1,623+ anti-A 2077 ± 262 3,110 ± 351 15,573 ± 691+ A.THanti-A.TL 3676 ± 798 6,063 ± 1,077 14,288 ± 1,486

Chimeric QT cells +Q spleen 3655 ± 309 6,420 ± 556 32,777 ± 384+ A.THanti-A.TL 2349 ± 228 3,492 ± 876 30,663 ± 4,142

Chimeric B1O.Q T cells were obtained as noted in legend to Table 1.Irradiated B1O.A spleen cells were added and the interaction betweenchimeric B1O.Q T cells and B1O.A APC was examined with anti-H-2aantiserum and ATH anti-A.TL antiserum. Controls to ensure speci-ficity of the A.TH anti-A.TL antiserum are shown in the last two lines.

right of I-E, this serum should only recognize determinants onB1O.A cells encoded by the I-At through I-Ek subregions. Chi-meric B1O.Q T cells responded to GAT when a source of re-sponder APC (B1O.A spleen cells) was added to the culture (Ta-ble 2). Addition of anti-A antiserum, which was capable ofreacting only with the B1O.A APC and not the respondingB1O.Q T cells, completely inhibited the GAT response as wasshown in Table 1. The GAT response of the chimeric B1O.Q Tcells was also inhibited by addition of the A.TH anti-A.TL anti-serum. Control experiments showed that the A.TH anti-A.TLantiserum had no effect on the GL4 proliferative response,which requires B1O.Q APC, confirming its specificity for B1O.AIa determinants. Thus, the data in Tables 1 and 2 suggest thatnonresponder T cells maturing in a responder environment be-come phenotypic responders by acquiring a receptor for re-sponder Ia molecules, and it is this capacity to recognize re-sponder Ia as self which determines the T cell's immuneresponse phenotype.

However, it was possible that other specificities in the anti-H-2 and A.TH anti-A.TL antisera besides anti-Ia antibodieswere responsible for the inhibitory effects. For example, it isconceivable that the anti-H-2a antiserum contains anti-T cell re-ceptor antibodies which act on the B1O.Q T cell receptor to in-hibit its interaction with responder B1O.A APC in the GAT re-sponse (13). To eliminate this and other possible explanationsfor the inhibitory effects ofthe antisera, a monoclonal anti-Ia. 17antibody was used. BlO.T(6R) -* (BlO.A X B1O.Q)F1 chimeraswere given T cell-depleted F1 spleen and bone marrow as asource of responder APC and immunized. Lymph node T cellswere isolated 8 days later. Any F1 cells surviving this procedurewere killed with anti-A antiserum plus complement. The chi-meric BlO.T(6R) T cells were found to be GL4 responders andGAT nonresponders (Table 3), but they made a substantial pro-liferative response to GAT when responder B1O.A APC wereadded in the form of irradiated spleen cells. The GAT responseofthe chimeric BlO.T(6R) T cells was virtually eliminated by theanti-Ia. 17 antibody directed against the responder B1O.A APCwhereas the GL4 response, which required H-24 restrictionelements, was unaffected. This result unequivocally establishesthat it is the Ia antigens on the APC that are being affected bythe anti-Ia antisera in these blocking experiments.

In the reciprocal experiment, B1O.A -- (B1O.A X BLO.Q)Flchimeras were given T cell-depleted F1 spleen and bone mar-

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Proc. Natl. Acad. Sci. USA 78 (1981) 517

Table 3. Monoclonal anti-Ia.17 inhibits BlO.A-restricted immune responses of chimeric BlO.T(6R) Tcells but not BlO.Q-restricted immune responses of chimeric B1O.A T cells

Cells Medium GAT GL4Chimeric BlO.T(6R) T cells 211 ± 81 398 ± 48 20,393 ± 1,983Chimeric BlO.T(6R) T cells + A

spleen 704 ± 88 15,865 ± 918 24,511 ± 1,727+ anti-Ia.17 monoclone 523 ± 61 1,081 ± 59 22,649 ± 2,119

Chimeric AT cells 1592 ± 312 20,888 ± 1,608 1,368 ± 40Chimeric AT cells + Q spleen 1684 ± 19 23,903 ± 2,947 22,783 ± 48+ anti-Ia.17 monoclone 1374 ± 9 1,745 ± 128 16,307 ± 307+ anti-Q antiserum 807 ± 210 21,963 ± 667 874 ± 235

B1O.A -. (B1O.A x B1O.Q)F1 and BlO.T(6R) -. (B1O.A x BlO.Q)Fl chimeras were given 108 T cell-de-pleted bone marrow and spleen cells intravenously and immunized in the hind footpads and base of tail;draining lymph nodes were harvested 8 days later. After passage through a nylon wool column, BlO.T(6R)-. F1 T cells were treated with anti-A antiserum plus complement to eliminate any residual F1 cells ad-ministered during immunization, and B1O.A -+ F1 T cells were treated with anti-Q antiserum plus com-plement. The resulting cells were "chimeric T(6R) T cells" and "chimeric B1O.A T cells," respectively.B1O.A irradiated spleen cells were added to chimeric BlO.T(6R) T cells and the interaction was blockedwith anti-I-Ak. B1O.Q irradiated spleen cells were added to chimeric B1O.A T cells and the interaction wasblocked with anti-I-Ak or anti-Q antiserum.

row cells at immunization as a source of GLbfresponder APC,and any remaining F1 cells were killed with anti-Q antiserumplus complement before assay. Chimeric B1O.A cells thus iso-lated were assayed and found to respond to GAT but not GL4(Table 3). When B1O.Q spleen cells were added as a source ofGL4b responder APC, the chimeric B1O.A T cells made a sub-stantial proliferative response to GL4. Addition of anti-Ia. 17 ata final concentration of 10% inhibited the GAT response almostcompletely but had little effect on the GL4 response. Thus, theinteraction between genotypic B1O.A T cells and B1O.Q APC togenerate a GL4 response was not inhibited by an anti-Ia anti-body specific for the T cell. The GL4 response was inhibited byanti-Q antiserum, showing again that only antibodies against theresponder APC can inhibit the Ir gene-controlled response.

DISCUSSION

The puzzle in I region control of immune responses has longbeen to relate the serologically detectable surface structuresthat are specified by the I region (Ia antigens) to the functionsthat map to the I region. The observations that I region-con-trolled T-cell proliferative responses (3) andT cell-dependent invitro antibody responses (14) could be inhibited by anti-Ia anti-sera and not by anti-K or anti-D antisera suggested that the Iaantigens might be the Ir gene products. Furthermore, subre-gion mapping revealed a complete concordance between Ia an-tigens and Ir genes (3, 4)-that is, Ir gene-controlled responsesmapping to I-A were inhibited by anti-Ia antisera with I-A-de-termined specificities and Ir gene-controlled responses map-ping to I-E/C were inhibited by anti-Ia antisera with I-E/C-de-termined specificities. Finally, in studies with (responder xnonresponder)F, mice, anti-Ia antisera specific for the re-sponder parental haplotype inhibited responses controlled by Irgenes of that haplotype, whereas antisera specific for the non-responder haplotype had little or no effect (3). This haplotypespecificity implied a phenotypic linkage between the Ir geneproduct and the Ia-bearing molecule at the cell surface, butwhich cell type was being blocked remained a matter for conjec-ture.A direct approach to the question ofwhich cell type is blocked

by anti-Ia antisera is the inhibition ofimmune functions involv-ing histoincompatible cells. In such assays, the antisera can bechosen to be selectively directed against either the APC or theresponding T cell. This was first attempted by Thomas et aL (15)in the guinea pig. These workers depleted strain 13 lympho-

cytes ofalloreactivity to strain 2 Ia molecules by culturing strain13 T cells with strain 2 macrophages in vitro, followed by elim-ination of the dividing cells by treatment with bromodeoxyuri-dine and light. The remaining strain 13 T cells could then inter-act with trinitrophenyl-modified strain 2 macrophages to mounta trinitrophenyl-specific proliferative response. This responsewas iphibited by antibody against the macrophage (13 anti-2antisera) but not by antibody against the T cell (2 anti-13 anti-sera). However, the interpretation of these data was somewhatclouded by the fact that anti-2 alloreactivity was not completelydepleted by the negative selection step; therefore, residual al-logeneic effects might have been responsible for yielding aber-rant results.

Nonetheless, the approach ofThomas et aL (15) appeared tobe the best strategy for addressing the question ifone could to-tally eliminate allogeneic effects. Therefore, we turned to theuse of"tetraparental" radiation-induced bone marrow chimeras[PI + P2-* (P1 x P2)Fj], whose lymphocytes had been shownby von Boehmer et aL (16) to be totally devoid of alloreactivitydirected against either parental donor haplotype. These chi-meras permitted an analysis of the interactions of histoincom-patible T cells and APC in reciprocal experiments in the absenceof any interference from allogeneic effects. Using this model,we have clearly demonstrated in this paper that anti-Ia antiseraact by blocking the APC from initiating a T-cell proliferative re-sponse. Anti-Ia sera directed against the T cell are without ef-fect.

The fact that the Ir gene-controlled T-cell response was se-lectively inhibited by a monoclonal antibody directed at a singledeterminant on a responder Ia molecule strongly suggests thatthe Ia molecule is the Ir gene product. Alternatively, it remainsa formal possibility that the Ia antigen and the Ir gene productare different structures but are so closely linked at the surface ofthe APC that binding to the Ia determinant sterically blocks anadjacent Ir gene product. Another possibility is that the anti-Iaantisera might induce a haplotype-specific suppression whichacts on any response using that Ia restriction element. Thesepossibilities cannot be ruled out by the techniques used here.Analysis ofI region mutations (17) and possibly DNA cloning ofthe I region may be required to exclude these less-likely possi-bilities.

Another important feature of the data presented here is thefinding that nonresponder stem cells are not discriminatedagainst in the chimera. We found no evidence for haplotypepreference in T-cell maturation in chimeras. In the B1O.A +

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518 Immunology: Longo and Schwartz

BlO.Q -+ (B1O.A X BLO.Q)Fj chimeras, if the responder stemcells had been preferentially selected to mature into responderT cells, one might have observed that the mixture of chimericBLO.A and BlO.Q T cells had made a higher GL4 response thanthe BLO.A T cells alone. Instead, it was observed that 4 x 105chimeric B1O.A plus B1O.Q T cells made about the same level ofGL4 response as 4 X 1 chimeric nonresponder B1O.A T cells(in Table 1 compare the GL# responses in lines 2 and 4). Simi-larly, the chimeric BlO.Q T cells, which are genotypically GATnonresponders, made about the same level of GAT response asthe mixture of BLO.A and BLO.Q T cells (Table 1, compare theGAT response in lines 2 and 8). Even for the non-Ir gene-con-trolled response to PPD, no haplotype preference was seen.The PPD response of the chimeric B1O.A T cells was blockedabout equally well by anti-A and anti-Q antisera as was the PPDresponse ofthe chimeric BLO.Q T cells. Thus, it appears that thsgenotype of the T cell has no predisposing effect upon the self-restriction repertoire it will develop in the thymus.

Finally, the experiments described in this paper examine themechanism of the phenotypic alteration of the T cell in radia-tion-induced bone marrow chimeras. Chimeric nonresponder Tcells maturing in a responder environment acquired the abil-ity to make Ir gene-controlled responses, but only when the an-tigen was presented on responder APC. In addition, the prolif-erative response of the chimeric nonresponder T cells wasinhibited by a monoclonal anti-Ia antibody directed against theresponder APC. These results suggest that the chimeric nonre-sponder T cells acquire the ability to respond by developing areceptor specific for responder Ia molecules. The implication isthat nonresponder T cells are not genetically defective becausethey can make Ir gene-controlled responses when they mature

in an environment that allows them to recognize responder Ia asself.

1. Shevach, E. M. & Rosenthal, A. S. (1973) J. Exp. Med. 138,1213-1229.

2. Yano, A., Schwartz, R. H. & Paul, W. E. (1978) Eur.J. Immunol 8,344-347.

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