Internal Complementarities in the Immune System: Regulation Ofthe Expression of Helper T-Cell Idiotypes

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Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4520-4523, July 1984 Immunology Internal complementarities in the immune system: Regulation of the expression of helper T-cell idiotypes (anti-,u suppression/helper-T-celI repertoire) CARLOS MARTINEZ-A.*t, PABLO PEREIRA*t, ROSA BERNABO, ANTONIO BANDEIRA*, EVA-LOTTA LARSSON*, PIERRE-ANDRE- CAZENAVE*, AND ANTONIO COUTINHO* *Department of Immunology, Pasteur Institute, Paris, France; and tDepartment of Immunology, Clinica Puerta de Hierro, Madrid, Spain Communicated by Baruj Benacerraf, April 4, 1984

ABSTRACT More than half of BALB/c helper T lympho- responses were induced with either LPS from Salmonella cytes specific for 2,4,6-trinitrophenyl (TNP)-modified synge- abortus equi (Difco) or specific helper T cells (see below) in neic spleen cells are inhibited in their proliferative responses to 0.2-ml cultures in microtiter plates (Falcon, Microtest II). antigen-presenting cells and in their cooperation with B lym- The culture medium was RPMI 1640 containing a growth- phocytes by monoclonal anti-idiotypic antibodies directed to a supporting, nonstimulatory selected batch of fetal calf serum TNP-binding BALB/c myeloma protein (MOPC 460). This in- (Flow Laboratories, no. 114) at 10%, 50 ,uM 2-mercapto- hibition is specific for anti-TNP-self helper cells of BALB/c ethanol, 10 mM Hepes, and antibiotics. origin and is controlled by IgCh-linked genes, as it is not ob- Hapten-Derivatized Cells. Spleen cell suspensions were served with CB.20 helper cells of the same specificity. In con- hapten derivatized with 2,4,6-trinitrophenyl (TNP), fluores- trast, anti-TNP-self helper cells prepared from BALB/c mice cein isothiocyanate (FITC), or 3-(p-sulfophenyldiazo)-4-hy- that were chronically suppressed with anti-tt chain antibodies droxyphenyl (SP) as described (12). and possessed no B lymphocytes were not inhibited by anti- Enriched Helper Cells. Cell lines with specificity for synge- idiotypic antibodies. We conclude that the B-cell antibody rep- neic TNP-, FITC- or SP-modified spleen cells were prepared ertoires contribute to the selection of the (idiotypic) T-helper- as described (12). Briefly, the draining lymph nodes of mice cell repertoires. primed with 50 x 106 hapten-derivatized syngeneic spleen cells in the base of the tail 4 days earlier were restimulated in The finding of antibody idiotypes on helper T cells has been vitro with the homologous antigen [haptenated spleen cells assumed to reflect the expression of immunoglobulin VH irradiated with 3300 rads (1 rad = 0.01 gray)]. These prepara- genes by all lymphocytes (1). Recently, however, molecular tive cultures of lymph node cells were fed and restimulated genetic approaches have excluded this possibility (2, 3), rais- weekly and used as a source of helper cells from 3 weeks ing interesting questions on the Ig allotype-linkage of idio- onwards. Proliferative responses of helper cells were studied typic determinants associated with T-cell specificities (4, 5). by coculturing 3 x 104 responding cells with 105 3300-rad- The "network" theory of the immune system considers lym- irradiated, hapten-coupled spleen cells and measuring tritiat- phocyte populations in a dynamic equilibrium of mutual idio- ed thymidine uptake after a 3-hr pulse with 1 IxCi (= 37 kBq) typic interactions continuously selecting for available clonal of [methyl-3H]thymidine (Radiochemical Centre, Amer- repertoires (6). These perspectives could contribute a solu- sham, England). Helper activity was measured by cocultur- tion for that paradox, as it could be assumed that "T-cell ing titrated numbers of helper cells (5 x 104 to 103) with 5 x idiotypes" were selected by the available B-cell and anti- 104 normal or hapten-derivatized spleen cells and measuring, body repertoire (7). We have now tested this hypothesis, 4 or 5 days later, the numbers of Ig-secreting cells in the comparing the ability of monoclonal anti-idiotypic antibodies staphylococcal protein A plaque assay (13). to inhibit specific T helper cells derived from normal or B- cell-deprived mice. The results indicate that the expression RESULTS of T-cell idiotypes is regulated by the B-lymphocyte compartment. Specific Inhibition of BALB/c Anti-TNP-Self Helper T Cells by Monoclonal F6(51) Antibodies. Helper T cells with speci- MATERIALS AND METHODS ficity for hapten-modified syngeneic cells, obtained by in vivo priming and repeated in vitro restimulation, are induced Mice. Mice of the strains BALB/c, CB.20, and C57BL/6, by hapten-derivatized presenting cells to extensive prolifera- bred at the Pasteur Institute, were used between 8 and 16 tion and, in turn, induce antigenic B cells into clonal expan- weeks of age. sion and polyclonal immunoglobulin secretion (12). Both re- B-Cell-Deprived Mice. These mice were obtained as de- sponses are restricted by the major histocompatibility com- scribed by Janeway et al. (8) by daily injection from birth plex and require direct cellular interaction (14). We have with rabbit anti-mouse IgM antiserum. Spleen and lymph screened lines of hapten-specific helper cells with monoclo- node cells from treated mice were individually tested by im- nal anti-idiotypic antibodies recognizing the corresponding munofluorescence (9) for the presence of immunoglobulin anti-hapten antibodies in the appropriate strains, in search of positive cells and for responsiveness to the B-cell mitogen a functional assay for idiotype-positive T cells. Our experi- lipopolysaccharide (LPS) in mass cultures (10) and in limit- mental system appeared suitable to this investigation, being dilution assays (11). Only those animals in which B lym- cause the cooperative responses are solely determined by phocytes were undetectable were used in these experiments. the specificity of the helper cells, and the responding B cells Lymphoid Cell Suspensions and Cultures. Lymphoid cell (potentially all) are not selected on the basis of clonal speci- suspensions were prepared as described (12). B-lymphocyte ficities. Furthermore, these protocols allow for the indepen-

The publication costs of this article were defrayed in part by page charge Abbreviations: FITC, fluorescein isothiocyanate; LPS, lipopolysac- payment. This article must therefore be hereby marked "advertisement" charide; SP, 3-(p-sulfophenyldiazo)-4-hydroxyphenyl; TNP, 2,4,6- in accordance with 18 U.S.C. §1734 solely to indicate this fact. trinitrophenyl; pfc, plaque-forming cells.

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dent quantitation of helper cell proliferation and ciooperative Table 1. Specificity and Ig allotype linkage of the F6(51) anti- B-cell responses in identical experimental conditiions. idiotype inhibition of anti-TNP BALB/c helper cells In the course of these experiments, we have identified Addition IgM % inhibition positive reactions between a monoclonal anti-i(diotype to Helper cell origin of F6(51) pfc/ of helper BALB/c anti-TNP antibodies (15) with helper ce]Ils specific and specificity antibodies culture activity for TNP-BALB/c spleen cells. This anti-idiotop)e, F6(51), was derived from BALB/c mice immunized with the TNP- BALB/c - 10,750 specific myeloma protein MOPC 460 and was shovwn to react anti-SP-BALB/c + 9,800 <10 specifically with a fraction of the anti-TNP antib)odies pro- BALB/c - 14,100 duced by every BALB/c mouse (15). As shown in Fig. 1, the anti-FITC BALB/c + 12,900 <10 titration of F6(51) antibodies into cultures of TNP'-BALB/c- BALB/c - 8,400 specific helper cells with haptenated spleen cells r)roduces a anti-TNP-BALB/c + 3,900 64 dose-dependent decrease in both helper cell prioliferation CB.20 - 5,700 and cooperative B-cell responses. This finding w' is fully re- anti-TNP-CB.20 + 6,100 producible -with a number of independently deriNved helper C57BL/6 - 4,900 cell lines repeatedly tested, maximal specific inhibiition being anti-TNP-C57BL/6 + 5,100 60-75%. The anti-idiotype inhibition was highly specific and Helper cell lines with specificity for syngeneic spleen cells deriva- Ig allotype linked. Thus, as shown in Table 1, F(6(51) anti- tized by TNP, FITC, or SP from the indicated strains were tested for bodies block up to 64% of the cooperative B-cell responses helper activity on the appropriate syngeneic derivatized spleen cells induced by "TNP-specific" helper cells, while havring no ef- in the presence or absence of F6(51) antibodies added at 15 ,gg/ml at fect on the responses induced by BALB/c helper cells spe- the start of the cultures. The table shows the day 4 IgM pfc respons- cific for SP- or FITC-derivatized syngeneic splieen cells. es observed and the inhibition calculated from these data. These experiments satisfactorily demonstrate the relatinn- ship between antigenic specificity and idiotype expression derivatized spleen cells from CB.20 mice, which are allotype by helper cells that should be expected if such idiotypes congenic with BALB/c mice. We conclude that the expres- were associated with clonally distributed receptors. More- sion of F6(51) idiotopes on TNP-specific helper T cells is over, as also shown in Table 1, the expression of F 6(51) idio- linked to immunoglobulin allotype and, consequently, that types by TNP-specific helper cells is controlled by genes these observations closely reproduce classical experiments linked to IgCh, the immunoglobulin heavy chain alllotype lo- on idiotype expression by T lymphocytes (1). In fact, the cus, because the F6(51) antibody has no effect inicoopera- stringent linkage to heavy chain locus of T-cell idiotype tive cultures containing anti-TNP helper cells and syngeneic expression has constituted the strongest argument for VH gene expression on T lymphocytes, because it excludes the possibility of mimicry between antigen and anti-idiotype as observed in several systems (16, 17). Helper Cells from Anti- g-Suppressed Mice Are Not Inhibit- ed by F6(51) Anti-Idiotypic Antibodies. The above observa- 12..S5 30 tions provided the experimental system necessary to the test of the hypothesis that T-cell idiotopes are regulatorily select- 0 ed by internal complementarities with antibodies and B cells. B-cell-deprived IBALB/c mice were produced by suppression with rabbit anti-,a antibodies, administered continuous- 0 ly from birth, as conventionally described (8). As shown in

L. Table 2, these mice lack B lymphocytes defined by surface expression of Ig and in functional assays of mitogen reactiv- ity. Ttus, less than 0.5% of spleen cells were stained by anti- 7.55. 18 Ig antibodies, and the number of LPS-reactive clonal precur- sors determined in limiting dilution was less than 1/200th of x that in normal age-matched untreated controls. X Helper T-cell lines with specificity for TNP-BALB/c were 2- produced from these anti-,-suppressed mice by immuniza- tion and repeated stimulation with TNP-derivatized spleen cells from normal, nonsuppressed BALB/c donors. The ef- fectiveness of the anti-,u suppression was monitored every

2.5 6 Table 2. Absence of B lymphocytes in anti-,u-suppressed mice LPS responses Limiting % Ig+ Mass culture dilution V.L Mice cells cpm pfc analysis 0.12 0.6 3 15 0 Normal 42 45,000 32,000 1/30 F6(51), ,ug/ml Anti-A-suppressed <0.5 <100 <100 <1/500 <0.5 <100 <100 ND FIG. 1. Specific anti-TNP BALB/c helper cells (after 4 weeks of <0.5 < 100 < 100 ND enrichment) were restimulated with irradiated TNP-derivatized BALB/c spleen cells, and the proliferative responses were mea- Spleen cells from normal or anti-,u-suppressed mice were tested sured on day 3 of culture. The same cell populations were used to for the presence of Ig-bearing B lymphocytes and LPS responsive- evaluate cooperative IgM plaque-forming cell (PFC) responses, ness as described (9-11). The results shown were obtained in the measured 4 days later. The cells were cultured either in the absence same experiment, which is representative of two others. In limiting or in the presence of the indicated concentrations of purified F6(51) dilution analysis, the numbers shown refer to clonal precursor fre- antibodies added at the start. quencies in total spleen cells. ND, not done. Downloaded by guest on September 28, 2021 4522 Immunology: Martinez-A. et al. Proc. NatL Acad Sci. USA 81 (1984)

75 mice. On the other hand, it could be argued that anti-pL treat- 0 ment had resulted in the activation of idiotype-specific suppressor cells that would inactivate competent helpers expressing that idiotype. This possibility was evaluated by E] studying the F6(51) antibody-mediated inhibition in mixtures I , of helper cells with the same specificity, produced in normal and anti-,p-suppressed mice. Results of this experiment are

n - shown in Table 3, and they demonstrate the lack of idiotype- specific suppressor activity in these cell populations. We S. li* . "o U conclude, therefore, that anti-/x-suppressed mice lack expression of the F6(51) idiotope on anti-TNP helper T cells. 0o - 6-° t' 25 DISCUSSION D;s. I fU c_.: To The nature of the mechanisms selecting available repertoires of lymphocytes is still the most immunological of all prob- lems. To analyze the role of B cells and antibodies on the helper cell repertoire we have taken advantage of the expres- 0o sion of a common idiotope by both B cells and helper cells 0.12 0.6 3 15 with the same nominal antigen specificity. Although T-cell F6(51), jug/ml idiotypes had been classically shown to be linked to the recent results from the mo- FIG. 2. Specific anti-TNP BALB/c helper cells were prepared immunoglobulin allotype locus, from adult (8-week-old), anti-,p-suppressed, B-cell-deprived lecular genetic analysis of T-cell clones have shown the lack BALB/c mice and were tested for proliferative responses and helper of VH gene rearrangement and expression on T cells. Regula- activity, in parallel with helper cells derived from normal mice. The tory interactions mediated by idiotypic complementarities indicated concentrations of F6(51) antibodies were added at the start between immunoglobulin and T cells could, however, oper- of the test cultures. Control, noninhibited responses were 32,640 ate in selecting T-cell populations expressing common poly- cpm and 11,400 pfc per culture for normal helper cells (e, m) and morphic antigenic determinants with immunoglobulins. This 15,450 cpm and 4200 pfc per culture for helper cells derived from hypothesis was directly tested by the ability of monoclonal anti-,u-suppressed mice (a, a). anti-idiotope antibodies to inhibit the activity of helper cells with the same nominal specificity. Thus, F6(51) anti-idiotope time when the immune lymph node donors were sacrificed. antibodies suppress anti-TNP helper cells from normal Of 12 animals, 8 were satisfactorily suppressed, such as BALB/c mice but not from those anti-IgM-treated and those shown in Table 2, and were used in these experiments, chronically suppressed donors. while the other 4 were discarded (numbers of splenic B lym- In our experimental system, in which the specificity is de- phocytes varying from 2.5% to 20%). No MOPC 460 idiotype termined solely at the level of the helper cells, the suppres- was detected in the serum of the suppressed mice as deter- sion observed cannot be ascribed to the responding B cells, mined in solid-phase assays using the F6(51) anti-idiotype because potentially all are able to respond. Furthermore, the antibody (not shown). "stimulator-responder" B cell populations are the same (i.e., As shown in Fig. 2, anti-pz-suppressed mice are fully com- from normal donors) both in cultures that are sensitive and in petent to generate helper cells with this specificity. Striking- cultures that are refractory to inhibition by anti-idiotypic ly, however, the titration of 1P6(51) antibodies to cooperative antibodies, which contain helper cells from normal or anti-,p- cultures completely fails to inhibit either helper cell prolif- suppressed donors, respectively. eration or helper cell-induced B cell responses. Since the These results demonstrate that, in the absence of a normal derivatized spleen cell populations are from normal BALB/c B-cell compartment, helper cells do not express idiotypic donors, these observations exclude that the anti-idiotype in- markers of antibodies with similar specificity. Since, in this hibition described above results from the interaction of the particular case, idiotype sharing is observed in normal mice, anti-idiotypiq antibodies with B cells or antibodies present in the present observations would suggest that idiotypic inter- the assay, and they make it likely that the anti-idiotype di- actions contribute to the selection of available T-cell reper- rectly interacts with responder T cells. toires. This could readily explain the Ig allotype linkage of These results could indicate absence of F6(51) idiotope "T-cell idiotypes" and the existing controversy between mo- expression by anti-TNP helper T cells in anti-it-suppressed lecular biologists and cellular immunologists on this ques- . , tion. Table 3. Absence of idiotype-specific suppressor activity in The present system, however, deserves a few comments: anti-v-suppressed mice (i) The inability to detect binding of the monoclonal F6(51) antibody to inhibitable helper cells by immunofluorescence Addition IgM (not shown) does not exclude binding at all, as it has recently of F6(51) pfc/ S II anti- Mice antibodies culture inhibition been described for low-affinity monoclonal anti-class bodies (19). (ii) The ability of anti-,u-suppressed mice to gen- Normal - 9700 erate hapten-modified specific helper cells described here + 3900 60 contrasts with reports on their failure to generate helper cells Anti-A-suppressed - 6300 specific for conventional soluble proteins (20) but agrees + 5900 <10 with others that have shown generation of helper, delayed- Normal + anti-ju-suppressed* - 7050 type hypersensitivity, and cytotoxic T-cell responses in such + 4200 41 mice (21). (iii) Our results are also in agreement with inde- Helper cell lines from normal or anti-IL-suppressed mice with pendent observations of altered idiotope expression in anti- specificity for syngeneic spleen cells derivatized with TNP were ,u-suppressed mice (21). prepared and tested as described for Fig. 1. It remains to be established whether such idiotypic selec- *One-to-one mixture of helper cells for normal and anti-IA-sup- tion is primarily thymic or peripheral, as well as what the pressed mice. possible ontogenic constraints of this process are. The an- Downloaded by guest on September 28, 2021 Immunology: Martinez-A. et aL Proc. Natl. Acad. Sci. USA 81 (1984) 4523

swers to these questions might help to solve the apparent 10. Andersson, J., Coutinho, A., Lernhardt, W. & Melchers, F. discrepancy between our observations and those of others (1977) Cell 10, 27-34. who have previously claimed that the expression of idio- 11. Andersson, J., Coutinho, A. & Melchers, F. (1977) J. Exp. types by T cells is antibody independent (18, 22, 23). Med. 145, 1511-1519. 12. Martinez-A., C., Coutinho, A., Bernabd, R. R., Augustin, A., Haas, W. & Pohlit, H. (1980) Eur. J. Immunol. 10, 403-410. We thank J. Badella for excellent help in the preparation of this 13. Gronowicz, E., Coutinho, A. & Melchers, F. (1976) Eur. J. manuscript. This work was partially supported by a grant from the Immunol. 6, 588-590. Fondo de Investigation de la Seguridad Social (Spain). 14. Martinez-A., C. & Coutinho, A. (1981) Nature (London) 290, 60-61. 15. Buttin, G., Le Guern, C., Phalente, L., Lim, E. C. C., Me- 1. Rajewsky, K. & Eichman, K. (1977) Contemp. Top. Immuno- drano, L. & Cazenave, P.-A. (1978) Curr. Top. Microbiol. Im- biol. 7, 69-112. munol. 81, 27-36. 2. Kraig, E., Kronenberg, M., Kapp, A. J., Pierce, C. W., 16. Sege, K. & Peterson, P. A. (1978) Proc. Natl. Acad. Sci. USA Abruzzini, A., Sorensen, C., Samelson, L., Schwartz, R. & 75, 2443-2447. Hood, L. (1983) J. Exp. Med. 158, 192-209. 17. Jerne, N. K., Roland, J. & Cazenave, P.-A. (1982) EMBO J. 1, 3. Marx, J. L. (1983) Science 221, 444-446. 243-247. 4. Binz, H., Wigzell, H. & Bazin, H. (1976) Nature (London) 264, 18. Julius, M., Cosenza, H. & Augustin, A. (1978) Eur. J. Immu- 639-640. nol. 8, 848-852. 5. Krawinkel, U., Cramer, M., Imanishi-Kari, T., Jack, R. S., 19. Layet, C., Le Bouteiller, P. P., Olive, D., Mishall, Z., Caillol, Rajewsky, K. & Makela, 0. (1977) Eur. J. Immunol. 8, 566- D., Kourilsky, F., Jordan, B. R. & Lemonier, F. A. (1984) 571. Eur. J. Immunol. 14, 99-102. 6. Jerne, N. K. (1974) Ann. Immunol. (Inst. Pasteur) 125 C, 373- 20. Ron, Y., De Baetselier, M., Gordon, J., Feldman, M. & Segal, 389. S. (1981) Eur. J. Immunol. 11, 964-970. 7. Rajewsky, K. & Takemori, T. (1983) Annu. Rev. Immunol. 1, 21. Sy, M.-S., Lowy, A., HayGlass, K., Janeway, C. A., Gurish, 569-607. M., Greene, M. I. & Benacerraf, B. (1984) Proc. NatI. Acad. 8. Janeway, C. A., Murgita, R. A., Weinbaum, F. I., Asofsky, Sci. USA 81, 3846-3850. R. & Wigzell, H. (1977) Proc. Natl. Acad. Sci. USA 74, 4582- 22. Krawinkel, K., Cramer, M., Kindred, B. & Rajewsky, K. 4586. (1979) Eur. J. Immunol. 9, 815-820. 9. Forni, L. (1979) in Immunological Methods, eds. J. Lefkovits 23. Weinberger, J. Z., Greene, M. J., Benacerraf, B. & Dorf, & B. Pernis (Academic, New York), pp. 151-167. M. E. (1979) J. Exp. Med. 149, 1336-1348. Downloaded by guest on September 28, 2021