Proc. Nati Acad. Sci. USA Vol. 78, No. 6, pp. 3809-3813, June 1981

A novel type of T-T interaction removes the requirement for I-B region in the H-2 complex (Ia /Ir /T-cell proliferation/suppression) CONSTANTIN N. BAXEVANIS, ZOLTAN A. NAGY, AND JAN KLEIN* Max-Planck-Institut fir Biologie, Abteilung Immungenetik, 7400 Tuibingen, Federal Republic of Germany Communicated by George Klein, February 26, 1981

ABSTRACT When tested in the in vitro T-cell proliferation MATERIALS AND METHODS assay, H-2a cells are nonresponders to lactate dehydrogenase B (LDH-B; L-lactate:NAD' oxidoreductase, EC 1.1.1.27) and to IgG2a myeloma protein. However, the cells can be converted into Mice. Mice were obtained from our colony at the Max- responders either by the addition to the culture ofmonoclonal anti- Planck-Institute for Biology. The strains and their alleles at H- Ia.m7 or by the removal from the culture of Lyt-2+ [T- 2 regions are given in Table 1. -associated alloantigen (Lyt)-2 positive] . Antigens. The ammonium sulphate precipitate of LDH-B4 In both instances, the responsiveness can be suppressed again by (Boehringer Mannheim) was extensively dialyzed against cul- the addition to the culture of monoclonal to I region- ture medium and stored at 4°C. Purified IgG2a myeloma pro- associated (Ia) molecules controlled by the I-A subregion. These teins UPC10 and RPC5 were obtained from Bionetics (Frank- data suggest that, in some H-2 haplotypes, the response to LDH- furt, Federal Republic of Germany) and stored at -70°C. B and IgG2a is the result of interaction between the I-A and I-E Antibodies. Hybridoma antibodies in ascites form were a gift subregions. The H-2l haplotype carries a responder allele at the from G. Hammerling (German Cancer Research Center, Hei- I-A subregion but the responsiveness ofH-2a cells is normally sup- delberg, Federal Republic of Germany). The following anti- pressed by T cells recognizing the in the context of the I- bodies were used: B15-124R4 (anti-Ia.m2), 13/18 (anti-Ia.m7), E molecules. When the recognition ofI-E molecules is blocked by B22-277Rl9 (anti-Ia.m8), 17-227R7 (anti-Ia.m5, recognizing an antiserum or when the cells capable of this recognition are re- Ia. 15), B22-24aRl (anti-H-2.m2), and H100-30/3 (anti-H-2.m5) moved, the H-2' cells become responders. These experiments (11). Antibody P47-42 (anti-Ia. m9, recognizing Ia. 13) was a gift demonstrate a nonresponder turned responder by antibody in- hibition. They also demonstrate that the postulate ofthei-B subre- from G. Gotze (Max-Planck-Institute for Biology, Tfibingen, gion is no longer necessary and provide additional evidence that Federal Republic of Germany). Nonspecific inhibitory sub- the Ia molecules are the products of the immune response (Ir) stances of low molecular weight occasionally present in ascites genes. fluids were removed by ultrafiltration with Amicon XM-1OOA filters (12). Lyt-2.2 (K564 pool III) alloantiserum [againstT-lym- The specificity ofantigen recognition by T cells is restricted by phocyte-associated alloantigen (Lyt)-2.2] was produced by im- genes in the major histocompatibility complex (MHC) (1). Two munizing (B6-Ly-2a X C3H/HeJ)F1 mice with C57BL/6 thy- recent lines ofevidence indicate that the immune response (Ir)- mocytes and was tested for specificity as described (13). phenomenon, that is the MHC-controlled difference in Immunization and Cell Cultures. Mice were injected sub- immune responsiveness to certain antigens (reviewed in ref. 2), cutaneously at the base of the tail with 50 ,ug of LDH-B or is a manifestation of this restriction. First, Ir-gene products The stan- have been shown to be expressed on the antigen-presenting cell myeloma proteins in complete Freund's adjuvant (14). but not on the responding , and they appear to function dard in vitro T-cell proliferation assay of Alkan (14) was used by providing the MHC context for recognition of the foreign with slight modifications as described (7). Antigen concentration antigen (3, 4). Second, with one exception, Ir loci ofthe MHC in the cultures was 15 ,ug/ml of LDH-B and 125 jig/ml ofmy- appear to be identical with those coding for plasma-membrane eloma protein. Control cultures contained concanavalin A I region-associated (Ia) molecules (5-8). The exception is the Ir (Wellcome, 25 yg per ml) or medium alone. Monoclonal anti- gene that maps in the so-called I-B subregion (here B region, bodies at the appropriate dilutions were included in the same for short) of the H-2 complex. The B region controls the re- volume (0.2 ml) of medium and were present throughout the sponse to at least two antigens, IgG2a myeloma protein (9) and culture period. Proliferation was measured by [3H]thymidine lactate dehydrogenase B (LDH-B; L-lactate:NAD' oxidoreduc- uptake on day 3 ofculture (7). All determinations were done in tase, EC 1. 1. 1.27) (10), but codes for no serologically detectable triplicate, and data were expressed as cpm ± SD. Significant membrane molecule. responses over background (i.e., cells without antigen) were In this communication we provide evidence that the B region calculated by using Student's t-test. The proliferation was me- is an illusion generated by a previously unrecognized form of diated by T cells as demonstrated by abrogation ofthe response interaction between two other regions ofthe H-2 complex, the after treatment of the lymph-node cells with Thy-1.2-specific A region and the E region. Thus, we remove the last objection antiserum and rabbit complement (data not shown). to the hypothesis in which the serologically detectable MHC molecules are the Ir-gene products. Abbreviations: Ia, I region-associated; Ir, immune response; LDH-B, lactate dehydrogenase B (L-lactate:NAD' oxidoreductase, EC 1.1.1.27); The publication costs ofthis article were defrayed in part by page charge Lyt, T-lymphocyte-associated alloantigen; MHC, major histocompati- payment. This article must therefore be hereby marked "advertise- bility complex. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 3809 Downloaded by guest on October 2, 2021 3810 Immunology: Baxevanis et al. Proc. Natl. Acad. Sci. USA 78 (1981)

Table 1. Proliferative T cell responses of standard and recombinant strains to LDH-B Alleles at H-2 regions Anti-LDH-B proliferative response H-2 Respondert Experiments, Strain haplotype K A B J E S D A cpm range* S.I. ranget status no. C57BL/6 b b b b b b b b 11,590-76,895 4.6-11.6 + 4 BO.D2 d d d d d d d d 20,042-70,502 4.7-16.5 + 3 B1O.GD g2 d d d b b b b 15,352-58,442 5.5-14.7 + 3 B1O.A a k k k k k d d -596-947 0.7-1.3 - 4 A.AL al k k k k k k d -1779-375 0.8-1.5 - 4 BlO.A(2R) h2 k k k k k d b -865-1,141 0.9-1.5 7 B1O.A(4R) h4 k k b b b b b 5,241-36,307 3.6-17.5 + 4 BlO.A(5R) i5 b b b k k d d 4,959-56,385 4.7-16.5 + 3 B1O.WB j j j j j j j j -162-587 0.9-1.2 - 4 BMO.P p p p p p p p p 3,566-32,160 6.2-25.2 + 3 B1O.Q q q q q q q q q 4,075-42,375 3.2-13.7 + 3 BlO.RIII r r r r r r r r 49,765 13.3 + 1 B1O.S s s s s s s s s 35,296 10.1 + 1 BlO.S(9R) t4 s 8 . k k d d 19,124 6.9 + 1 B1O.HTT t3 s s s s k k d 26,046-28,425 8.4-9.8 + 2 BlO.SM v v v v v v v v 60,000 11.7 + 1 * cpm of cultures with antigen-cpm of cultures without antigen, lowest and highest value, respectively. t Lowest and highest stimulation index (ratio of cpm in cultures with/without antigen). * Responder (+): significant proliferative responses in all experiments. Nonresponder (-): no significant response over background. § Putative alleles of recombinant strains used for definition of the I-B subregion are boxed. Treatment of Cells with Lyt-2.2-Specific Antiserum and Proliferative T-Cell Responses of Standard and Recombi- Complement. One volume ofcell suspension (1 x 107 cells per nant H-2 Haplotypes to LDH-B. The antibody response of the ml) was incubated with 1 vol of antiserum or normal mouse inbred mouse strains to LDH-B is controlled by a dominant Jr serum (1:5 dilution) at 4°C for 45 min. Thereafter, 1 vol ofrabbit gene that maps to the B region of the H-2 complex (10). We on vitro complement (1:4) was added, and incubation was continued at tested whether the same Ir-gene effect is exerted the in 37°C for 1 hr (13). proliferative response of T cells to LDH-B. The effect of antigen concentration on the proliferative re- RESULTS sponse ofprimed lymph-node cells is shown by the experiments Rationale. The original definition ofthe B region was based in Fig. 1. Optimal responses were obtained at 15 ,ug of LDH- on two recombinants, H-25 and H-2h4, both derived from an B per ml in culture. Unprimed cells of responder strains (such H-2a/H-2b heterozygote. The H-2a haplotype is a low responder as C57BL/6) or primed cells of nonresponder strains (such as to both myeloma protein and LDH-B, whereas H-2b and the B1O.A) gave no response over the whole range ofantigen doses two recombinant haplotypes are high responders (9, 10). These tested. observations formally delineate the B region in the following The experiments performed to determine the genetic control manner: ofT-cell proliferation to LDH-B are summarized in Table 1. The H-2 haplotypes a, al, and h2, classified as nonresponders in H-2"5 Kb Ab BHR jk Dd terms ofantibody production, proved also to be nonresponders in the T-cell proliferation assay. Furthermore, 10 other H-2 JK H-2a Kk A' BLR Dd haplotypes were also responders at both the antibody and the T-cell level. The recombinant strains used to localize the rel- BH H-2b Kb Ab jb Db evant Jr gene to the B region responded identically in both as-

H-2h4: Kk Ak BHR jb Db

However, one can also explain the data without evoking the 4 existence of a new region. Because H-2 haplotypes b and h4 do not express any cell-surface E molecules (15), one can assume that they are high responders to IgG2a and LDH-B because x they carry high-responder alleles at the A locus. The H-2a hap- 2 1 lotype, which also carries the Ak allele but (in contrast to H-2b V K and H-2"4) does express E molecules, must be a low responder O.44N a=. I because either (i) in the presence of cell-surface E molecules oI . . --.0.. , , ...... %.. the response occurs, for some reason, preferentially through the E molecules (the Ek being a low-responder allele) or (ii) the Ek 0 3.9 15.6 62.5 250 0 3.9 15.6 62.5 250 is a suppressor allele that blocks the response normally involv- Antigen concentration in culture, Ag/ml ing the A molecules. FIG. 1. Effect of antigen concentration on the proliferative re- If our reasoning were correct, one should be able to turn the sponse of lymph-node cells to LDH-B. Primed (W) and unprimed (0) H-2a into a high-responder haplotype by blocking the E region- C57BL/6 (responder) cells and primed B1O.A (o, non-responder) cells controlled response. We tested this prediction by blocking the were cultured in the presence of LDH-B at the concentrations indi- T-cell proliferative response to LDH-B and IgG2a with mono- cated. Vertical bars represent ±SD (not shown, where too small to be clonal Ia antibodies. represented graphically). Downloaded by guest on October 2, 2021 Immunology: Baxevanis et al. Proc. Natl. Acad. Sci. USA 78 (1981) 3811

says. However, we have found one discrepancy in the respon- C57BL/6 BlO.D2 siveness ofH-2i strains. The strain C3H.JK tested for antibody production to LDH-B was a responder (16), whereas strain 0T500 50 120000TT200 12 BIO.WB was a consistent nonresponder in the T-cell prolifer- ation assay (Table 1). Although the reason for this discrepancy is not known, we should point out here that (i) the mechanism ofunresponsiveness in the B1O.WB strain is different from that in other nonresponder strains (Fig. 1) and (ii) the H-2J haplotype 6 of strains C3H.JK and B1O.WB are of different origin. on Influence of Monoclonal Ia Antibodies the T-Cell Prolif- r erative Response to LDH-B. We have demonstrated that T-cell responses controlled by Ir genes in the A region are blocked x o 5000 2500 1250---- 0- 2000---- 1000--- 500 by monoclonal antibodies against the A molecule, whereas re- sponses under dual Ir gene control are only inhibited by an antibody against the E molecule (7). Because the localization of the Ir-LDH-B gene in the B region may be a phenotypic manifestation of two counteracting Ir genes in the A and E re- gion, respectively, we tested the effect ofmonoclonal antibodies against the A or E molecule on the proliferative response to LDH-B. The proliferative response ofH-2 haplotypes b, d, g2, h4, i5, p, and q was completely inhibited with monoclonal antibodies against the A molecule expressed by the strain tested (Figs. 2 and 3). No inhibition was observed with Ia. m7-specific antibody recognizing a determinant on the a chain of the E molecule. Reciprocal of antibody dilution

FIG. 3. Inhibition ofT-cell proliferative responses to LDH-B with monoclonal Ia antibodies directed against the I-A molecule. The an- tibodies added to cultures were anti-Ia.m2 (m), anti-Ia.m5 (co), anti- Ia.m7 (A), anti-Ia.m8 (C), anti-Ia.m9 (o), and anti-H-2.m5 (oj). , Relevant antigen is expressed by the responding strain; ----, relevant antigen is not expressed. In control cultures, proliferation was mea- sured in the presence (0) or absence (o) of antigen with no antibody added. Vertical bars represent ±SD.

Monoclonal antibodies against la determinants not expressed or H-2 determinants expressed by the responding strain had no effect on proliferation. These results indicate that the prolif- erative response to LDH-B occurs through the A molecule. The 0C striking finding was that the nonresponder H-2 haplotypes a,

x al, and h2, which were used to define the B region, all turned to responders when the E molecule was blocked with Ia.m7 antibody (Fig. 2). The proliferative response obtained in the presence of Ia. m7 anitibody could be blocked by the addition ofantibodies against the A molecule. However, antibodies against the A molecule alone had no influence on the response. A similar but only par- tial interference ofthe E molecule was observed with the anti- LDH-B response ofBlO.A(5R) cells. T-cell proliferation by this responder strain to LDH-B was significantly enhanced when Ia.m7 antibody was added to the cultures (Fig. 2). Thus, cell- surface expression of the E molecule can interfere to different degrees with the anti-LDH-B response governed by the A mol- ecule. Alternative mechanisms of unresponsiveness are also 0 10,000 5000 2500 0-10,000 5000 2500 0 10,000 5000 2500 possible as exemplified by the B1O.WB strain, which could not Reciprocal of antibody dilution be turned to responder by either the Ta. m7 (Fig. 2) or the Ia.m5 antibody (data not shown). FIG. 2. Effect of on re- monoclonal Ia antibodies the anti-LDH-B Proliferative to sponse ofresponder [BlO.A(5R), B1O.A(4R)] and nonresponder [BlO.A, Response LDH-B by Nonresponder Cells A.AL, BlO.A(2R), and BlO.WB] strains. The antibodies added to cul- Treated with Anti-Lyt-2.2 and Complement. To clarify the tures were against Ia.m2 (n), Ia.m5 (o), Ia.m7 (A), Ia.m8 (-), Ia.m7 plus mechanism of unresponsiveness associated with the E mole- Ia.m2 (A), Ia.m7 plus Ia.m5 (h), and H-2.m2 (v). -, Relevant antigen cule, we tested the possible role of functional T-cell subpopu- is expressed bythe strain tested; - ---, relevant antigen is not expressed. lations in the response to LDH-B. As shown by the represent- In control cultures, proliferation was measured in the presence (o) or ative experiment in Fig. 4, primed cells ofa nonresponder strain absence (o) ofantigen, with no antibody added. Vertical bars represent gave a strong response to LDH-B treatment -+-SD. H-2.m2 is a private determinant of H-2Db; H-2.m5 is a class I proliferative after determinant encoded by H-2 haplotypes b, k, p, q, r, s, u, and v. Ia.m9 with anti-Lyt-2.2 antiserum plus complement. The response of is a determinant encoded by I-AP and I-Aq alleles. For distribution of these cells was not influenced by Ia.m7 antibody but was other determinants, see the footnote to Table 2. strongly inhibited with antibodies against Ia.m2 and Ia.m5 Downloaded by guest on October 2, 2021 3812 Immunology: Baxevanis et al. Proc. Natl. Acad. Sci. USA 78 (1981)

against NMS + C aLyt-2.2 + C to responder by treatment with Ia.m7 antibody directed the E molecule (Table 2). Removal of Lyt-2 cells also turned a mechanism of 4 8 haplotype h2 to responder (Table 2). Thus, the Ir gene control exerted on the responses to IgG2a and LDH- B appears to be identical. A L DISCUSSION x Because the original definition ofthe B region was based on the u 21 4 determination ofantibody responses in vivo (9, 10), whereas our data were obtained by measuring T-cell proliferation in vitro, we should first consider how the two types ofassay relate to each i other. First, we should point out that the H-2-linked control k a of most antibody responses, including that to LDH-B, occurs i5 through helper T cells (2, 10), and the same T cells appear to 0 10,000 5000 2500 0 10,000 5000 2500 proliferate in response to an antigen in vitro (17, 18). Second, Reciprocal of antibody dilution there is a correlation between the strain-distribution patterns ofresponsiveness in vivo and in vitro. In the case ofthe immune FIG. 4. (Right) Primed B1M.A(2R) cells become LDH-B-responders response to LDH-B, our in vitro typing matches (with one ex- plus rabbit comple- after treatment with Lyt-2.2-specific antiserum ception) the in vivo testing by Melchers et al. (10, 16). The one ment (C): effect ofmonoclonal Ia antibodies on the response. (Left) Cells strain C3H.JK is an interme- were pretreated with normal mouse serum (NMS) plus C. The anti- exception is the H-2i haplotype; bodies added to cultures were anti-Ia.m2 (o), anti.Ia.m5 (o), and anti- diate responder in vivo (16), whereas we found strain B10.WB Ia.m7 (A). Controls consisted ofcultures without antibody in the pres- to be a nonresponder in vitro. Because these strains are of in- ence (e) or absence (o) ofantigen and cultures with Ia.m7 antibody in dependent origin, differences either in H-2- or in non-H-2- the absence of antigen (o). Vertical bars represent ±SD. linked genes may account for the discrepancy (19-21). For the purpose of this study, the crucial fact is that there is a perfect (present on the A molecule). Thus, the proliferation is mediated match between the in vivo and in vitro responsiveness ofstrains by Lyt-1 cells, whereas the lack of response is associated with involved in the original definition of the B region-that is, the presence of Lyt-2 cells. This result strongly suggests, al- strains carrying H-2 haplotypes a, b, h4, and i5. We conclude, though it does not formally prove, that the observed unrespon- therefore, that the interpretations based on the in vitro data are siveness is due to suppressor T-cells generated by recognition also applicable to the in vivo observations. of LDH-B in the context of the E molecule. We propose that the postulate of a separate B region is no Proliferative T-Cell Responses to IgG2a Myeloma Proteins. longer necessary to explain the pattern of LDH-B responsive- Lymph-node cells from mice primed with the IgG2a myeloma ness. The interpretation that we offer instead is this. We assume for the low responsiveness is protein UPC10 gave significant proliferative responses when that the basis Ir-gene-controlled a failure of a T cell to be stimulated by a particular combination challenged with the same or another IgG2a myeloma protein of antigen and an MHC molecule. In the case of LDH-B, this RPC5 in vitro (data not shown). Thus the response, similar to can arise in at least two ways. One way is exemplified to be failure the antibody response against MOPC173 (9), appeared by the B10.WB strain carrying the H-2i haplotype. Here, the on the immunoglobulin directed against allotypic determinants haplotype probably contains a low-responder allele at the A lo- responses were at 125 molecule. Peak proliferative obtained ,ug cus so that there is no MHC molecule in the context of which of myeloma protein per ml of culture. The strain distribution the antigen could be recognized by the T cell; the E locus seems of responsiveness was identical with that of the antibody re- to have no influence on the recognition. The second way is ex- sponse to MOPC173, that is, it corresponded to a B-region-con- emplified by the H-2a haplotype of the B10.A strain. Here the trolled pattern of responsiveness (ref. 9; Table 2 and unpub- haplotype carries a high-response allele at the A locus, but the lished data). The proliferative response to IgG2a was influenced allele is prevented from exerting its positive effect by the E lo- by monoclonal Ia antibodies in the same manner as the anti- cus. When the effect ofthe E locus is neutralized, the H-2a cells LDH-B response. Thus, the proliferation by responder H-2 turn from low to high responders. The neutralization can be haplotypes h4 and i5 was blocked by antibodies against the A achieved in at least two ways: (i) by coating the cells with an- molecule, whereas the nonresponder haplotype h2 was turned tibodies directed against the cell surface E molecules or (ii) by

Table 2. Influence of monoclonal Ia antibodies on T-cell proliferative response to the IgG2a myeloma protein, UPC10 Determi- Proliferative responset in cultures supplemented with* H-2 nants* con- Pretreat- a-Ia.m7 a-Ia.m7 haplo trolled by ment of and and Strain type I-A I-E cells - a-Ia.m7 a-Ia.m2 a-Ia.m5 a-Ia.m2 a-Ia.m5 a-Ia.m8 B10.A(2R) h2 m2, m5 m7 630 (1.3) 23,340(12.6) 4,060(3.0) 4,800(3.8) 800(1.4) - _ B10.A(4R) h4 m2, m5 0 26,500(12.0) 23,850(10.5) -250(0.9) -200(0.9) B1O.A(5R) i5 m8, m5 m7 12,300 (5.1) 55,500(19.5) - 10,500(4.5) -450(0.8) -90(1.0) B10.A(2R) h2 m2, m5 m7 NMS + C 1,057 (1.4) 21,870(10.7) 4,297(2.9) 4,545(3.0) 2,092(1.9) B10.A(2R) h2 m2, m5 m7 aLyt-2.2 + C 21,712(10.6) 19,800 (9.8) - 2,565(2.1) 3,735(2.7) * Only Ia determinants detected by the monoclonal antibodies are shown. Ia.m2 is a private determinant ofI-Ak; Ia.m8 is a determinant shared by I-Ab and I-Ad; Ia.m5 is a determinant shared by H-2 haplotypes b, d, and k; and the Ia.m7 determinant is an E. determinant present in all strains that express this chain. t Expressed as A cpm with stimulation index in parentheses (see footnotes to Table 1). t The final dilution of monoclonal Ia antibodies in cultures was 1:2500 for Ia.m2 and Ia.m7 and 1:1250 for Ia.m5 and Ia.m8. Downloaded by guest on October 2, 2021 Immunology: Baxevanis et al. -Proc. Natl. Acad. Sci. USA 78 (1981) 3813 removing Lyt-2' cells from the culture. (The cells proliferating 1. Zinkernagel, R. M. & Doherty, P. C. (1974) Nature (London) in response to the antigen are ofthe Lyt-1+ Lyt-2- type.) These 248, 701-702. by postulating that LDH-B 2. Benacerraf, B. & Germain, R. N. (1978) Immunol. Rev. 38, 70- two observations can be explained 119. and the Ek molecule are recognized by suppressorTcells, which 3. Schwartz, R. H., Yano, A., Stimpfling, J. H. & Paul, W. E. then prevent proliferation ofT cells recognizing LDH-B in con- (1979)J. Exp. Med. 149, 40-57. text of the Ak molecule. 4. Longo, D. L. & Schwartz, R. H. (1980)J. Exp. Med. 151, 1452- The observation that the B10.A(4R) strain carries a high-re- 1467. sponderH-2 haplotype is consistent withour interpretation: this 5. Shevach, E. M., Paul, W. E. & Green, I. (1972)J. Exp. Med. cell-surface E molecules (15) and, 136, 1207-1221. strain does not express any 6. Schwartz, R. H., David, C. S., Dorf, M. E., Benacerraf, B. & therefore, the A molecule is not obstructed from manifesting Paul, W. E. (1978) Proc. Nati. Acad. Sci. USA 75, 2387-2391. its high responder potential. 7. Baxevanis, C. N., Wernet, D., Nagy, Z. A., Maurer, P. H. & The assumption that the suppression of the response in the Klein, J. (1980) Immunogenetics 11, 617-628. H-2a-bearing strains involves both the E and the A molecules 8. Lerner, E. A., Matis, L. A., Janeway, C. A. Jr., Jones, P. P., the BLO.A(5R) strain. The H-2'5- Schwartz, R. H. & Murphy, D. B. (1980)J. Exp. Med. 152, 1085- is supported by the typing of 1101. bearing cells respond to the LDH-B by proliferation but the 9. Lieberman, R., Paul, W. E., Humphrey, W., Jr. & Stimpfling, proliferation is enhanced by the addition of Ia.m7 antibodies. J. H. (1972) J. Exp. Med. 136, 1231-1240. Here, apparently, the interaction between the A and E loci is 10. Melchers, I., Rajewsky, K. & Shreffler, D. C. (1973) Eur.J. Im- such that partial response is allowed, but the full potential of munol. 3, 754-761. 11. Lemke, H., Hammerling, G. J. & Hammerling, U. (1979) Im- the response materializes only when the interference of the E munol. Rev. 47, 175-206. locus is completely abolished. The ability ofthe E molecule to 12. Klein, J., Chiang, C. L. & Wakeland, E. K. (1977) immunoge- generate suppression is clearly allele-specific, because the pres- netics 5, 445-451. ence of the Ed or EP allele does not influence anti-LDH-B 13. Vidovic, D., Jureti6, A., Nagy, Z. A. & Klein, J. (1981) Eur. J. responses. Immunol., in press. remain to be answered, one fact 14. Alkan, S. S. (1978) Eur. J. Immunol. 8, 112-118. Although many questions 15. Jones, P. P., Murphy, D. B. & McDevitt, H. 0. (1978)J. Exp. is clearly established by our data: there is no need to postulate Med. 148, 925-933. an extra B region to explain the response pattern of H-2 hap- 16. Melchers, I. & Rajewsky, K. (1975) Eur.J. Immunol.5, 753-759. lotypes a, b, h4, and i5 to LDH-B and IgG2a. This pattern is 17. Schwartz, R. H. & Paul, W. E. (1976)J. Exp. Med. 143, 529-540. fully explained by the participation in the response ofthe A and 18. Kapp, J. A., Ananeo, B. A. & Clevinger, B. L. (1980) J. Exp. E regions, the existence ofwhich is firmly established. On the Med. 152, 235-240; B region from 19. Falkenberg, F. W., Sulica, A., Shearer, G. M., Mozes, E. & basis of these results we propose to remove the Sela, M. (1974) Cell. Immunol. 12, 271-279. the H-2 map. 20. Wiener, E. & Bandieri, E. (1974) Eur. J. Immunol. 4, 457-463. We thank Drs. G. Hammerling and D. Gotze for monoclonal anti- 21. Suzuki, K., Fathman, C. G. & Tomasi, T. B., Jr. (1979) J. Im- bodies. This work was supported in part by Grant Wa 139/10/AI.5 from munol. 123, 1530-1533. the Deutsche Forschungsgemeinschaft. Downloaded by guest on October 2, 2021