Proc. Nati. Acad. Sci. USA Vol. 86, pp. 10069-10073, December 1989 Immunology Costimulatory signal provided by a B-lymphoblastoid cell line and its Ia-negative variant (antigen-presenting cells/mixed lymphocyte reaction/monoclonal antibody/T-celi activation) HANS REISER AND BARUJ BENACERRAF Department of Pathology, Harvard Medical School, and Division of Lymphocyte Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115 Contributed by Baruj Benacerraf, September 13, 1989

ABSTRACT We have analyzed the requirements of highly Our results indicate that both M12 and M12.C3 cells can purified, resting murine CD41 T lymphocytes for activation provide costimulatory function very efficiently and that the mediated by the lectin Con A and by monoclonal antibodies costimulator is expressed constitutively on the cell mem- against the CD3 and Thy-I molecules. Our results indicate that brane. Our experiments also show interesting differences in both the Ia-positive B-lymphoblastoid cell line M12 and its autoreactivity between this B-lymphoblastoid cell line and Ia-negative variant M12.C3 can provide the costimulatory normal Ia-bearing spleen cells. Finally, we show that this activity necessary for these activation pathways. The costim- system can be used in a simple screening assay to detect ulatory function is preserved upon fixation with paraformal- interesting monoclonal antibodies (mAbs) that affect cell dehyde, indicating that the costimulatory molecule(s) is (are) function. The characterization of some of these differentia- constitutively expressed on the cell surface. Our experiments tion antigens concerned with T-cell activation will be re- also point to interesting differences between the M12 cell line ported elsewhere. and syngeneic Ia-positive antigen-presenting cells in generating a syngeneic mixed lymphocyte reaction. Finally, we show that the CD4' T cell-M12.C3 cell interaction can be used to screen MATERIALS AND METHODS for interesting monoclonal antibodies that affect cell function. Animals. Mice and Armenian hamsters were purchased Physiologic T-cell activation depends on cognate interactions from The Jackson Laboratory and the Harvard School of of the T cell with an antigen-presenting cell (APC) (1, 2). Public Health, respectively, and maintained in accordance Available evidence suggests that antigens get internalized by with the guidelines ofthe Committees on Animals ofHarvard the APC, processed, and reexpressed on the APC surface (3, Medical School and of the Dana-Farber Cancer Institute. 4). The specificity of T-cell activation is imparted by a Antibodies. The following mAbs were used in this study: clonotypic T-cell receptor (TCR) that recognizes the proc- mAb MK-D6 = anti-I-Ad (29); mAb M5/114 = anti-I-A essed antigenic peptide in the context of proteins encoded anti-I-Edk (30); mAb 10-2.16 = anti-I-Ak (31); mAb M1/42 = within the major histocompatibility complex (MHC) (1, 5, 6). antiL-H-2 (all -haplotypes) (32); mAb 116-13.1 = anti-Lyt-2.1 Besides the TCR-MHC interaction, several other molec- (33); mAb ADH4 =-anti-Lyt-2.2 (34); mAb 145-2C11 = ular pairs seem to participate in T cell-APC interactions. (i) anti-CD3 (35); and mAb HK 2.1 = anti-Thy-1 (36). Normal heterogenous T lymphocytes or T-cell clones require Immunofluorescence and Fluorescence-Activated Cell Sorter costimulatory growth factors for their activation, which are Fluorocytometry. Cells were analyzed by indirect immunoflu- also provided by the APC (7). (ii) The TCR is noncovalently orescence and flow fluorocytometry as described (13). associated with a set of nonvariable proteins, termed T3 Cell Cultures. M12 (27), M12.C3 (28), M12.C3.F6 (37), and (CD3) (8-11). (iii) Work from our (12-14), as well as other (15, P3X63-Ag.653 cells (38), were passaged in Dulbecco's mod- 16), laboratories has shown that phosphatidylinositol-linked ified Eagle's medium supplemented as described (14). Sple- proteins can trigger an activation signal in T lymphocytes. (iv) nocytes were depleted of erythrocytes by treatment with The expression of the CD4 and CD8 molecules correlates Tris NH4Cl. T cells were enriched by nylon wool fraction- with the class of MHC protein to which the T cell is restricted ation (39). CD4' T cells from BALB/c (H-2d, Lyt-2.2) or (17, 18). The CD8-MHC class I and CD4-MHC class II pairs AKR (H-2k, Lyt-2.1) mice were purified by twofold treatment may play a role in cell-cell adhesions (19), as well as in signal with a mixture of mAbs (M5/114 plus ADH4 for BALB/c, transduction (20, 21). Similarly, the molecular pairs CD2/ M5/114 plus 10-2.16 plus 116-13.1 for AKR) and low-toxicity LFA-3 (22, 23), LFA-1/ICAM-1 (24, 25), and LFA- rabbit complement (Cedarlane Laboratories, Hornby, ON, 1/ICAM-2 (26) could serve both functions. It should be noted Canada). Microcultures were set up in duplicates in 96-well that the exact role of any of the above accessory molecules plates. Briefly, 2-5 x 105 T cells were cultured with or in T-cell activation and their precise topographical and func- B in 0.2 ml tional relationship to the TCR has not been determined. without 105 cells (M12, M12.C3, or M12.C3.F6) Furthermore, there may be more molecules involved in of RPMI 1640 medium (Irvine Scientific), supplemented with lymphocyte interactions and/or lymphocyte activation that 10o fetal calf serum, 4 mM L-glutamine, 10 mM Hepes, 50 are currently unknown. ,uM 2-mercaptoethanol, antibiotics, and nonessential amino Here we report the results of an analysis ofthe interactions acids. The precise culture constituents are described in the of purified CD4+ T cells with the B-lymphoblastoid cell line respective experimental protocols. After 20-24 hr, 0.1 ml of M12 (27) and its Ia-negative variant M12.C3 (28) in syngeneic culture supernatant was removed, irradiated (8000 rads; 1 rad mixed lymphocyte reactions (MLR) as well as in several = 0.01 Gy), and assayed for interleukin 2 content on the HT-2 pathways of antibody- and lectin-mediated T-cell activation. indicator cell line (12).

The publication costs of this article were defrayed in part by page charge Abbreviations: APC, antigen-presenting cell; mAb, monoclonal an- payment. This article must therefore be hereby marked "advertisement" tibody; MHC, major histocompatibility complex; MLR, mixed lym- in accordance with 18 U.S.C. §1734 solely to indicate this fact. phocyte reaction; TCR, T-cell receptor. 10069 Downloaded by guest on October 1, 2021 10070 Immunology: Reiser and Benacerraf Proc. Natl. Acad. Sci. USA 86 (1989) RESULTS Table 1. Accessory help provided by M12 and M12.C3 cells The B-Lymphoblastoid Cell Line M12 and Its Variant Cell line Stimulus cpm M12.C3 Can Provide Costimulatory Function for the Activa- - - 362 tion of Resting CD4+ T Lymphocytes. We chose the response Con A 1,127 of purified CD4+ T cells to the B-lymphoblastoid I-A+ I-E+ HK2.1 361 cell line M12 and its corresponding I-A- I-E- variant M12.C3 145-2C11 405 as model system. The latter two lines differ only in respect to M12 13,750 the surface expression of the MHC class II gene products. M12 Con A 121,125 The M12.C3 line is lacking an I-Ad transcript and thus cannot M12 HK2.1 50,020 express intact I-A molecules; furthermore, it expresses I-Ed M12 145-2C11 133,725 molecules only in an intracellular compartment (28). Note M12.C3 420 that the I-A- I-E- phenotype of M12.C3 cells is not affected M12.C3 Con A 149,125 by treatment with Con A (Fig. 1 Upper). To test the useful- M12.C3 HK2.1 24,317 ness of these cell lines we initially analyzed the autoreactive M12.C3 145-2C11 78,780 response of purified CD4+ class II-restricted T cells to M12 Microcultures were set up as detailed with 2 x 105 purified CD4' and M12.C3 cells in a lymphokine assay. As expected, the T cells and with or without 1 x 105 M12 and M12.C3 cells. Where T-cell response to self depends on the presence of Ia mole- indicated, Con A (1 jug/ml), or anti-Thy-1 mAb HK2.1 (1:8 super- cules and is completely abrogated when the M12.C3 cell line natant dilution) or anti-CD3 mAb 145-2C11 (1:10,000 supernatant is used instead of M12 (Fig. 1 Lower). To confirm that the dilution) was added to the cultures. After 20 hr, an aliquot of each M12.C3 cell line is functional, we analyzed M12.C3.F6, a culture was harvested and assayed for lymphokine content with the subclone of M12.C3, that had been transfected with the I-Aa HT-2 indicator cell line. and I-Ak genes and thus expresses I-Ak on the cell surface (ref. 37; Fig. 1 Upper). Clearly this line can support the either cell line. The results of two representative experiments lymphokine production of AKR (H-2k) T cells in response to with M12 and M12.C3 are shown in Table 1 and Fig. 2. Clearly, MHC class II protein (Fig. 1 Lower). both cell lines provide accessory cell function very efficiently. To test M12 and M12.C3 cells for costimulatory function, The Costimulatory Molecule(s) Is (Are) Constitutively Ex- normal CD4+ T lymphocytes were incubated with Con A, with pressed on the Cell Membrane of M12 and M12.C3 Cells. anti-CD3 mAb, or with anti-Thy-1 mAb in the presence of Having established that both M12 and M12.C3 cells could help activate resting CD4' T cells, we examined whether the MK-ED6 M5/t14 10-2 16 Ml142 B-cell lines needed to be activated to provide this costimu- latory function. Previous work in a different system by Weaver et al. (40) had shown that splenic B cells required activation to help activate T-cell clones. To investigate this question, we studied the ability of ------r - - paraformaldehyde-fixed M12 or M12.C3 cells to provide LK~~~ _---- costimulatory function in our system. Table 2 shows that

- EHK21 BALBfc : 145-2C11 CM 2 2 .. Y!2C3~ ~ ~ M12I...... III 0 1 2 3 0 1 2 3 cpm X1-4 cpm x 10 4 FIG. 1. Phenotype of the M12 and M12.C3 lines. (Upper) Un- treated M12, M12.C3, M12.C3.F6 cells or Con A-treated M12.C3 a 41 cells were analyzed by indirect immunofluorescence and fluores- cence-activated cell sorter fluorocytometry as described with the following mAbs: MK-D6 (anti-I-Ad), M5/114 (anti-l-Abdq, anti- 2 I-Ed k), 10-2.16 (anti-I-Ak), and M1/42 [anti-H-2 (all haplotypes)], and fluorescein isothiocyanate-goat anti-mouse IgG, crossreactive with rat IgG. Fluorescence was quantitated on a FACSscan flow cytometer (Becton Dickinson), and data are displayed as histograms iL of the logarithm of relative fluorescence intensity (x axis) versus linear cell number (y axis). Five thousand cells were analyzed per sample. Background fluorescence (thin line) represents staining with the second antibody only and is superimposed with the specific mAb staining in several panels. (Lower) Response ofCD4+ T cells to MHC oL class II antigens on M12 or M12.C3.F6 cells. CD4+ T cells from M12 M12 C3 BALB/c (H-2d, Lyt-2.2) or AKR (H-2k, Lyt-2.1) mice were purified by nylon wool separation and twofold treatment with a mixture of FIG. 2. Accessory help provided by M12 and M12.C3 cells in the mAbs. Microcultures were set up in duplicates in 96-well plates as response to purified activating mAbs. Microcultures were set up as detailed in text. Briefly, 5 x 105 T cells were cultured with or without described with 2 x 105 T cells, 1 x 105 M12 or M12.C3 cells, and 1 x 105 B cells (M12, M12.C3, or M12.C3.F6) in 0.2 ml of supple- purified mAb HK 2.1 (anti-Thy-1 mAb; 10 ,g/ml) or mAb 145-2C11 mented RPMI 1640 medium. After 20-24 hr, 0.1 ml of culture (anti-CD3 mAb; 250 ng/ml), where indicated. Titration experiments supernatant was removed, irradiated, and assayed for interleukin 2 had previously shown that the anti-CD3 reagent is stimulatory at content on the HT-2 indicator cell line. much lower concentration compared with the anti-Thy-1 mAb. Downloaded by guest on October 1, 2021 Immunology: Reiser and Benacerraf Proc. Natl. Acad. Sci. USA 86 (1989) 10071 Table 2. Preservation of the costimulatory activity of M12 and Table 4. Cocultures with Ia' spleen cells and M12.C3 cells as M12.C3 cells upon fixation with paraformaldehyde stimulators in syngeneic MLR Exp. Cell line Con A (1 gg/ml) cpm Con A Group T Cells Stimulator cells (1 cpm 1 319 tlg/ml) 1,055 I + - 100 M12 (n) 56,145 II + + 178 M12 (n) 69,380 III + 5 x 104 M12 - 68,672 M12 (f) 320 IV + 5 x 104 SC - 86 M12 (f) 80,630 V + 5 x 104 SC + 42,015 M12.C3 (n) 594 VI - 5 x 104 SC + 146 M12.C3 (n) 73,490 VII + 1 x 105 SC - 138 M12.C3 (f) 276 VIII + 1 x l05 SC + 73,820 M12.C3 (f) 54,515 IX - 1x105 SC + 255 2 876 X + 5 x 104 M12.C3 - 232 631 XI + 5 x 104 M12.C3 + 53,355 M12 (f) 2,890 XII + 1 x 105 M12.C3 - 297 M12 (f) 61,122 XIII + 1 x 105 M12.C3 + 69,910 M12.C3 (f) 2,128 XIV + 5 x 104 SC + - 309 M12.C3 (f) 97,436 5 x 104 M12.C3 XV + 1 x 105 SC + - 476 Cultures were constructed with 2 x 105 purified CD4' T cells and 1 x 105 live (n) or fixed (f) M12 or M12.C3 cells. In experiment 1, an lx 105 M12.C3 aliquot of 0.1 ml of culture supernatant was removed after 20 hr and Microcultures were constructed with the indicated number of assayed for lymphokine content on the HT-2 cell line. In experiment accessory cells [T cell-depleted spleen cells (SC) or M12.C3 cells] 2, T-cell proliferation was assayed by adding 1 ,uCi (1 Ci = 37 GBq) and 2 x 105 purified CD4+ T cells, except for groups VI and IX, of [3H]thymidine during the last 5 hr of a 48-hr incubation period. where no T cells were added. After 20 hr, aliquots of each culture were removed and assayed on the HT-2 cell line. both paraformaldehyde-fixed M12 and M12.C3 cells could help Con A-induced T-cell activation as measured by lym- pression of the putative costimulatory molecule. Two results phokine production and T-cell proliferation. argue against this possibility. (i) The same normal spleen cells Analysis of the Difference Between Normal Spleen Cells and could provide a costimulatory signal for Con A-mediated M12 Cells in Stimulatng a Syngeneic MLR. The high degree of T-cell activation (Table 3). It should be noted that the autoreactivity toward the Ia molecule seen in our system observed Con A responses are not due to a contamination of contrasts with that reported by other investigators who used the stimulator preparation with T cells, as these cells do not normal Ia-bearing splenic APCs instead of B-cell lines (41). We respond to Con A in the absence ofexogenously added T cells therefore compared these populations ourselves. Table 3 (ex- (Table 3, experiment 2; Table 4, groups VI and IX). (ii) The periment 1) shows that M12 cells stimulated the activation of addition of M12.C3 cells to a culture containing CD4+ T cells CD4' T cells, as expected. In contrast, normal splenic APCs, and la+ spleen cells did not reconstitute a syngeneic MLR even at a five times higher cell concentration, stimulated only (Table 4, group XIV and XV; data not shown). Control a marginal syngeneic MLR. Similar results were observed at all experiments include a positive syngeneic MLR towards M12 stimulator cell concentrations tested (data not shown). cells (Table 4, group III). Thus, based on these data, we Because of the known radiosensitivity of B cells, we also conclude that the absence of MLR stimulation by normal performed comparative experiments with nonirradiated T- spleen cells is not due to a lack of expression of the costim- cells as cell-depleted spleen stimulators (Table 3, experiment molecule. the cell do not 2; Table 4). These experiments gave qualitatively identical ulatory Also, spleen preparations results, pointing to a true difference between normal APCs appear to contain a subset of inhibitory cells, as we did not and M12 cells as stimulators in our assay. find any evidence of suppression of the M12-mediated MLR We next investigated whether the failure of normal splenic by the addition of BALB/c spleen cells (data not shown). APCs to support a syngeneic MLR was due to an underex- Generation of B-Cell Hybridomas That Inhibit the Interac- tions of CD4+ T lymphocytes with M12.C3 Cells. As a means Table 3. Comparison of normal Ia' spleen cells and M12 of identifying functional APC cell-surface antigens involved cells as stimulators in the interaction between T cells and the B-cell lines in our Exp. T Cells Stimulators Con A (1 ug/ml) cpm system, we attempted to produce mAbs that would affect that interaction. For this purpose, Armenian hamsters were re- 1 + - 679 peatedly injected i.p. with M12.C3 cells. After several rounds + + 855 of immunization, animals were sacrificed, and their spleen + 5x105SC - 2,021 cells were fused to cells. + 5 x 105 SC + 64,430 P3X63-Ag8.653 myeloma Hybrid- oma culture supernatants were tested for their ability to + 1 x 105 M12 - 44,276 2 + 238 inhibit anti-CD3-induced and M12.C3-dependent T-cell acti- + + 421 vation. Several distinct inhibitory mAbs were obtained, + 5 x 105 SC - 2,721 demonstrating the feasibility ofthis approach. Two examples + 5 x 105 SC + 52,890 are shown in Fig. 3. Both mAb 3-8B7 and mAb 5-8A10 can - 5 x 105 SC + 855 almost completely block the response of CD4+ T cells to + 1 x 105 M12 - 46,488 anti-CD3 antibody. A detailed characterization of the anti- gens recognized by these antibodies will be reported else- Microcultures were constructed with or without 2 X 105 purified where; however, a preliminary analysis of their expression CD4+ T cells and the indicated number of accessory cells [M12 or spleen cells (SC)]. Unfractionated irradiated spleen cells were used demonstrates that, while both of the antigens are expressed in experiment 1; T-cell-depleted nonirradiated spleen cells were used on the M12.C3 cell used in the immunizations, only the in experiment 2. After 20 hr, an aliquot of each culture was removed 5-8A10 but not the 3-8B7 antigen is expressed on CD4' T- and assayed for lymphokine content on the HT-2 cell line. cells (Fig. 4). Clearly, this pattern of expression maps the Downloaded by guest on October 1, 2021 10072 Immunology: Reiser and Benacerraf Proc. Natl. Acad. Sci. USA 86 (1989)

FIG. 3. Inhibition of the anti-CD3/M12.C3- Z.. dependent activation of resting CD41 T cells by mAbs 3-8B7 (A) and 5-8A10 (B). Microcultures were set up with 2 x 105 CD4' T cells and 1 x 105 M12.C3 cells, the indicated amount of anti- CD3 mAb 145-2C11 and either a 1:10 superna- tant dilution of control mAb (M1/42 = anti-H-2, /45-2CI (/lsupernatant d/lu/,bn2 4) or mAb 3-8B7 (A, *) or mAb 5-8A10 (B, v). inhibitory effect of mAb 3-8B7 during CD4' T cell-M12.C3 and its Ia-negative variant and in contrast to normal heter- interactions to the side of the B-lymphoblastoid cell line. ogenous B cells (40), the costimulatory signal is constitutively expressed on the cell surface and does not require prior cell activation. Recently, Nabavi et al. (37) expressed a truncated DISCUSSION form of the I-Ak molecule, lacking the carboxyl-terminal Activation of resting CD41 T lymphocytes requires the amino acids, in M12.C3 cells. The resulting transfectants occupation of at least one cell-surface receptor [e.g., the showed a dramatically decreased ability to induce interleukin TCR-CD3 complex (35), Thy-1 (15), an Ly-6-linked protein 2 production by some autoreactive T-T hybridomas, al- (12, 13, 16)] and the presence of at least one additional though the basis for this phenomenon remains to be deter- costimulatory signal derived from an APC (7). Here we report mined. Given our data on paraformaldehyde-fixed M12.C3 that both the B-lymphoblastoid cell line M12 and its Ia- cells, we feel that the results by Nabavi et al. (37) can hardly negative variant M12.C3 can provide the accessory cell be explained by the absence ofthe costimulatory signal on the function necessary for the activation of resting CD4' T cells. surface ofthe APC. In the system described by Schwartz and An analysis of T-cell activation mediated by the lectin Con A his collaborators (7), presentation of antigen by 1-ethyl- or by stimulatory mAbs against the Thy-1 molecule or against 3-(3-dimethylaminopropyl) carbodiimide-fixed APCs to Th-1 the CD3 component of the T-cell antigen receptor complex clones results in a state ofunresponsiveness, presumably due hereby gave qualitatively similar results. Our results are to a lack of costimulatory factor. This process can be consistent with previous experiments by Hunig (42) and by abrogated, however, by the addition of allogeneic spleen Malek et al. (43), who have shown that several murine cells, which has lead Mueller et al. (7) to suggest that the Ia-negative cell lines, including a fibroblastoid cell line and a costimulatory signal may be transmitted in the absence of a T-cell tumor, can provide a costimulatory function, although stable TCR-Ia-peptide interaction. This interpretation is the basis for this phenomenon was not defined. We have supported by our findings. taken these experiments one step further by actually com- An arralysis-of T-cell clones has suggested that the CD4+ paring an Ia-negative variant cell line to its wild-type parent T lymphocytes can be, subdivided into interleukin 2 (Th-1) and by demonstrating that the metabolically fixed B cell lines and interleukin 4 (Th-2) producers (44). More recent exper- M12 and M12.C3 can also provide accessory cell function. iments have raised the possibility that the costimulator for Thus, at least in the case of this B-lymphoblastoid cell line Th-1 clones is interleukin-1, whereas that of Th-2 clones is a hitherto unidentified molecule (45). Although the precise A B nature of the lymphokine(s) produced in our system remains to be determined, estimates of a precursor ratio of Th-1 and Th-2 cells of 500:1 among splenic CD4+ T cells suggest that the predominant lymphokine produced in our system is interleukin 2 (46). Consistent with this assumption, we have seen only a limited effect of interleukin 1 in our system (data not shown). A very intriguing aspect of the system described in this E C report is the high degree of reactivity toward the syngeneic Ia ~~~~~~~CD molecule, which is not observed when normal spleen cells are used as APCs (Table 3). Our results suggest that the lack of normal splenocytes to support such a response is not due to a lack ofcostimulator, as these cells do provide accessory cell function when Con A is added as a stimulator to the cultures and as cocultivation with M12.C3 cells as a source of co- stimulator does not reconstitute the syngeneic MLR (Table 4). The possibility that the costimulator needs to be present on the same cell as the Ta molecule-i.e., does not work in Log relative fluorescence intensity trans-seems unlikely given the reconstitution experiments by Schwartz and his coworkers (47). Formally, either one of FIG. 4. Expression of the determinants recognized by the mAbs the following hypotheses could explain our data. (i) The M12 3-8B7 and 5-8A10 on purified CD4+ T lymphocytes and on M12.C3 cell line could express a stimulatory protein on its cell surface cells. Purified CD4+ T cells (A and B) and M12.C3 cells (C and D) were analyzed by indirect immunofluorescence and flow fluorocy- that is not present on normal spleen cells. However, to tometry using the mAbs 3-8B7 (A and C) and 5-8A10 (B and D). Data explain the data obtained in the cocultivation experiment, are displayed as described in the legend to Fig. 1. The purity of the one would also have to assume the absence of this molecule CD4+ population used in A and B was confirmed by analysis of the from M12.C3 cells. (ii) Our data could be interpreted in the CD4 and CD8 markers. context of recent findings by Marrack and Kappler (48), who Downloaded by guest on October 1, 2021 Immunology: Reiser and Benacerraf Proc. Natl. Acad. Sci. USA 86 (1989) 10073 have shown that alloreactive V,817a' T-T hybridomas do 14. Reiser, H., Oettgen H., Yeh, E. T. H., Terhorst, C., Low, respond to the I-E molecule on B cell lines but not on thymic M. G., Benacerraf, B. & Rock, K. L. (1986) Cell 47, 365-370. cells or on the macrophage cell line P388D1. These 15. Gunter, K. C., Malek, T. R. & Shevach, E. M. (1984) J. Exp. epithelial Med. 159, 716-730. authors have proposed that a B-cell line-specific peptide 16. Malek, T. R., Ortega, G., Chan, C., Kroczek, R. & Shevach, could associate with the la molecule to give a stimulatory E. M. (1986) J. Exp. Med. 164, 709-722. Ia-antigen complex. Our data thus raise the possibility that in 17. Swain, S. L. (1983) Immunol. Rev. 74, 129-142. our system a particular, possibly tumor-specific, Ia-antigen 18. Meuer, S. C., Schlossman, S. F. & Reinherz, E. L. (1983) complex expressed on M12 cells stimulates a subset ofT cells Proc. Natl. Acad. Sci. USA 79, 4395-4399. a particular family. 19. Doyle, C. & Strominger, J. L. (1987) Nature (London) 330, bearing a T-cell receptor belonging to 256-259. Further studies are necessary to evaluate this problem. 20. Hunig, T. (1984) J. Exp. Med. 159, 551-558. Finally, it is also of interest, that the MLR is virtually 21. Bank, I. & Chess, L. (1985) J. Exp. Med. 162, 1294-1303. abolished upon fixation of M12 cells with paraformaldehyde. 22. Howard, F. D., Ledbetter, J. A., Wong, J., Bieber, C. P., A number of molecules on the cell surfaces of both the T Stinson, E. B. & Herzenberg, L. A. (1981) J. Immunol. 126, cell and the APC participate during T cell-APC interactions, 2117-2122. reason to believe that the list of 23. Meuer, S. C., Hussey, R. E., Fabbi, M., Fox, D., Acuto, O., and at present there is no Fitzgerald, K. A., Hodgdon, J. C., Protentis, J. P., Schloss- functional molecules identified so far is complete. We thus man, S. F. & Reinherz, E. L. (1984) Cell 36, 897-906. made use of our system in an attempt to identify functionally 24. Davignon, D., Martz, E., Reynolds, T., Kurzinger, K. & relevant antigens on the APC surface. For this purpose we Springer, T. A. (1981) Proc. Natl. Acad. Sci. USA 78, 4535- immunized Armenian hamsters with M12.C3 cells. It should 4539. be noted that such an immunization protocol could a priori 25. Pircher, H., Groscurth, P., Baumhutter, S., Aguet, M., Zinker- nagel, R. M. & Hengartner, H. (1986) Eur. J. Immunol. 16, give rise to mAbs recognizing a number of hitherto uniden- 172-181. tified murine antigens, including the costimulator for Th-1 26. Staunton, D. E., Dustin, M. L. & Springer, T. A. (1989) Na- clones as well as the murine homologue of human LFA-3. ture (London) 339, 61-64. Using this approach, we have derived two hamster mAbs 27. Kim, K. J., Kanellopoulos-Langevin, C., Merwin, R. M., with distinct tissue distribution that block the CD4' T cell- Sachs, D. H. & Asofsky, R. (1979) J. Immunol. 122, 549-554. M12.C3 interaction. A detailed characterization of the anti- 28. Glimcher, L. H., McKean, D. J., Choi, E. & Seidman, J. G. antibodies will be reported else- (1985) J. Immunol. 135, 3542-3550. gens recognized by these 29. Kappler, J. W., Skidmore, B., White, J. & Marrack, P. (1981) where (unpublished work), but it can already be noted that J. Exp. Med. 153, 1198-1214. these antibodies are not simply directed against known 30. Bhattacharya, A., Dorf, M. E. & Springer, T. A. (1981) J. murine adhesion molecules such as LFA-1 and ICAM-1 (data Immunol. 127, 2488-2495. not shown). Thus the immunization/screening protocol de- 31. Oi, V. T., Jones, P. P., Goding, J. W., Herzenberg, L. A. & scribed in this report allows for the generation of previously Herzenberg, L. A. (1978) Curr. Top. Microbiol. Immunol. 81, 115-129. undetected mAbs with interesting functional properties. 32. Stallcup, K. C., Springer, T. A. & Mescher, M. F. (1981) J. Immunol. 127, 923-930. We are grateful to Dr. Laurie Glimcher, to Dr. Jeff Bluestone, and 33. Shen, F. W. (1981) in Monoclonal Antibodies and T Cell to Dr. Frank Fitch for reagents and to Dr. Kenneth L. Rock for Hybridomas, eds. Hammerling, G. J., Hammerling, U. & discussions and for critically reviewing this manuscript. We espe- Kearney, J. F. (Elsevier, Amsterdam), pp. 25-31. cially acknowledge the help of Mrs. Colette F. Gramm and of Mr. 34. Gottlieb, P. D., Marshak-Rothstein, A., Auditore-Hargreaves, Leonard Colarusso with these experiments and thank Mary Jane K., Berkoben, D. B., August, D. A., Rosche, R. M. & Bene- Tawa for her assistance in the preparation of this manuscript. H.R. detto, J. D. (1980) Immunogenetics 10, 545-555. is the recipient of an Investigator Award from the Cancer Research 35. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. & Bluestone, Institute. J. A. (1987) Proc. Natl. Acad. Sci. USA 84, 1374-1378. 36. Lancki, D. W., Ma, D. I., Havran, W. L. & Fitch, F. W. 1. Rosenthal A. S. & Shevach, E. M. (1973) J. Exp. Med. 138, (1984), Immunol. Rev. 81, 65-94. 1194-1212. 37. Nabavi, N., Ghogawala, Z., Myer, A., Griffith, I. J., Wade, 2. Unanue, E. R. & Allen, P. M. (1987) Science 236, 551-557. W. F., Chen, Z. Z., McKean, D. J. & Glimcher, L. (1989) J. 3. Ziegler, K. & Unanue, E. R. (1981) J. Immunol. 127, 1869- Immunol. 142, 1444-1447. 38. Kearney, J. F., Radbruch, A., Liesegang, B. & Rajewsky, K. 1875. (1979) J. Immunol. 123, 1548-1550. 4. Allen, P. M. & Unanue, E. R. (1984) J. Immunol. 132, 1077- 39. Julius, M. H., Simpson, E. & Herzenberg, L. A. (1973) Eur. J. 1079. Immunol. 3, 645-649. 5. Benacerraf, B. (1978) J. Immunol. 120, 1809-1812. 40. Weaver, C. T., Hawfylowicz, C. M. & Unanue, E. R. (1988) 6. Yague, J., White, J., Coleclough, C., Kappler, J., Palmer, E. & Proc. Natl. Acad. Sci. USA 85, 8181-8185. Marrack, P. (1985) Cell 42, 81-87. 41. Lattime, E. C., Gillis, S., Pecoraro, G. & Stutman, 0. (1982) J. 7. Mueller, D. L., Jenkins, M. K. & Schwartz, R. H. (1989) Immunol. 128, 480-485. Annu. Rev. Immunol. 7, 445-480. 42. Hunig, T. (1984) Eur. J. Immunol. 14, 483-489. 8. Reinherz, E. L., Meuer, S., Fitzgerald, K. A., Hussey, R. E., 43. Malek, T., Schmidt, J. A. & Shevach, E. M. (1985) J. Immu- Levine, H. & Schlossman, S. F. (1982) Cell 30, 735-743. nol. 134, 2405-2413. 9. Meuer, S. C., Acuto, O., Hussey, R. E., Hodgdon, J. C., 44. Mosmann, T. R. & Coffman, R. L. (1989) Annu. Rev. Immu- Fitzgerald, K. A., Schlossman, S. F. & Reinherz, E. L. (1983) nol. 7, 145-173. Nature (London) 303, 808-810. 45. Greenbaum, L. A., Horowitz, J. B., Woods, A., Pasqualini, 10. Borst, J., Prendiville, M. A. & Terhorst, C. (1982) J. Immunol. T., Reich, E. P. & Bottomly, K. (1988) J. Immunol. 140, 128, 1560-1565. 1555-1560. 11. Samelson, L. E., Harford, J. B. & Klausner, R. D. (1985) Cell 46. Powers, G. D., Abbas, A. & Miller, R. A. (1988) J. Immunol. 43, 223-231. 140, 3352-3357. 12. Rock, K. L., Yeh, E. T. H., Gramm, C. F., Haber, S. I., 47. Jenkins, M. K., Ashwell, J. D. & Schwartz, R. H. (1988) J. Reiser, H. & Benacerraf, B. (1986) J. Exp. Med. 163, 315-333. Immunol. 140, 3324-3330. 13. Reiser, H., Yeh, E. T. H., Gramm, C. F., Benacerraf, B. & 48. Marrack, P. & Kappler, J. (1988) Nature (London) 332, 840- Rock, K. L. (1986) Proc. Nati. Acad. Sci. USA 83, 2954-2958. 843. Downloaded by guest on October 1, 2021