Proc. Natl. Acad. Sci. USAVol. 88, pp. 6575-6579, August 1991Immunology

B-cell surface antigen B7 provides a costimulatory signal thatinduces T cells to proliferate and secrete interleukin 2CLAUDE D. GIMMI*t, GORDON J. FREEMAN*, JOHN G. GRIBBEN*, KANJI SUGITA*, ARNOLD S. FREEDMAN*,CHIKAO MORIMOTO*, AND LEE M. NADLER*Departments of *Medicine and *Pathology, Division of Tumor Immunology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115

Communicated by Baruj Benacerraf, April 15, 1991

ABSTRACT Occupancy of the T-cell receptor complexdoes not appear to be a sufficient stimulus to induce a T-cell-mediated immune response. Increasing evidence suggests thatcognate cell-cell interaction between an activated T cell and anantigen-presenting cell may provide such a stimulus. A candi-date T-cell surface molecule for this costimulatory signal is theT-cell-restricted CD28 antigen. Following crosslinking withanti-CD28 mAb, suboptimally stimulated CD28' T cells showincreased proliferation and markedly increased secretion of asubset of lymphokines. Recently, the B-cell surface activationantigen B7 was shown to be a natural ligand for the CD28molecule, and both B7 and CD28 are members of the immu-noglobulin superfamily. Here we report that B7-transfectedCHO cells can induce suboptimally activated CD28+ T cells toproliferate and secrete high levels of interleukin 2. The re-sponse is identical whether T cells are submitogenically stim-ulated with either phorbol myristate acetate or anti-CD3 toactivate the T cells. This response is specific and can be totallyabrogated with anti-B7 monoclonal antibody. As has previ-ously been observed for anti-CD28 monoclonal antibody, B7ligation induced secretion of interleukin 2 but not interleukin4. We have previously demonstrated that B7 expression isrestricted to activated B lymphocytes and interferon V-acti-vated monocytes. Since these two cellular populations areinvolved in antigen presentation as well as cognate interactionwith T lymphocytes, B7 is likely to represent a central costim-ulatory signal that is capable of amplifying an immune re-sponse.

Although engagement of the T-cell receptor complex (TCR)by antigen in the context of proteins encoded by the majorhistocompatibility complex (MHC) is essential for the initialstages of T-cell activation, it does not appear to be sufficientto induce all the events that accompany T-cell activation (1).Studies suggest that costimulation through additional T-cellsurface molecules, independent of the TCR, leads to en-hanced proliferation and cytokine production (2-4). There-fore, signals that trigger these accessory molecules are likelyto be essential for the generation of an immune response andtheir dysregulation may be responsible for immune-mediateddisease states (1, 5, 6).On the T cell, a candidate to receive this accessory cell

contact signal is the 44-kDa homodimeric T-cell surfaceprotein CD28 (7-9). This T-cell-restricted molecule, which isa member of the immunoglobulin superfamily, is expressedon 95% of CD4+ cells, on 50% of CD8+ cells, and onthymocytes that coexpress CD4 and CD8 (4, 10-12). Follow-ing suboptimal activation of T cells with anti-CD3 monoclo-nal antibody (mAb) (4, 13), anti-CD2, or phorbol 12-myristate13-acetate (PMA) (14, 15), crosslinking ofCD28 by anti-CD28mAb results in enhanced T-cell proliferation (2, 8, 15-17) andgreatly augments synthesis of multiple lymphokines (18). The

magnitude ofenhancement oflymphokine synthesis resultingfrom engagement of the CD28 pathway distinguishes it fromother cell surface ligand pairs between T cells and antigen-presenting cells (APCs) such as LFA-1/ICAM-1 (19-23) andCD2/LFA-3 (24-26). The observed increase in lymphokineproduction by anti-CD28 crosslinking results initially fromstabilization oflymphokine mRNAs (27, 28) and later from anincrease in transcription (29). Only a subset of T-cell-derivedlymphokines are induced by the CD28 pathway, includinginterleukin 2 (IL-2), interferon y, tumor necrosis factor a, andgranulocyte/macrophage colony-stimulating factor but notIL-4 (27). This observation suggests that either alternativesignals or subpopulations of T cells may be necessary toinduce IL-4 synthesis (1).The natural ligand for the CD28 molecule has recently been

shown to be the B-cell activation antigen B7 (30). Heterotypicbinding of cell lines transfected with CD28 and B7 andspecific inhibition of this binding by either anti-CD28 oranti-B7 mAbs have convincingly demonstrated that B7 is aCD28 ligand and this interaction can mediate T-B cell adhe-sion (30). The B7 molecule is a 44- to 54-kDa glycoprotein thatis also a member of the immunoglobulin superfamily (31). B7expression is extremely restricted in that B7 is transientlyexpressed on activated B cells and interferon y-treated mono-cytes (32-34). Since these two cellular populations are in-volved in antigen presentation as well as cognate interactionwith T lymphocytes, the interaction of B7 and CD28 is likelyto represent a costimulatory signaling pathway. In the pres-ent report, we demonstrate that the B7 molecule is not onlyan adhesion molecule for CD28 but also capable of upregu-lating proliferation and markedly enhancing lymphokine pro-duction in suboptimally stimulated CD28+ T cells.

MATERIALS AND METHODSCells. Human peripheral blood mononuclear cells were

isolated from buffy coats obtained by leukopheresis ofhealthy donors. After density gradient centrifugation the cellswere further purified by depletion ofadherent cells on plastic.Residual B cells and monocytes were depleted by passagethrough nylon wool. The CD28+ subset of T cells wasenriched by separation from the reciprocal subset ofCD11b+T cells (2, 12, 35), residual B cells, and monocytes by twotreatments with complement lysis utilizing anti-3B8 (CD56),anti-Mol (CD11b), anti-Mo2 (CD14), and anti-B1 (CD20)mAbs. The efficiency of the purification process was ana-lyzed in each case by indirect cell immunofluorescence andflow cytometry (Coulter EPICS flow cytometer) using T3(CD3) and 4B10 (CD28) mAbs and fluorescein isothiocy-

Abbreviations: TCR, T-cell receptor complex; MHC, major histo-compatibility complex; mAb, monoclonal antibody; PMA, phorbol12-myristate 13-acetate; IL, interleukin; APC, antigen-presentingcell.tTo whom reprint requests should be addressed at: Division ofTumor Immunology, Dana-Farber Cancer Institute, Mayer 730, 44Binney Street, Boston, MA 02115.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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anate-labeled goat anti-mouse immunoglobulin (Tago). Thefinal T-cell preparation was >90%o CD3' and >88% CD28+in each case when compared with staining with an isotype-identical unreactive control antibody. Examination of smearsstained for nonspecific esterase (a-naphthyl acetate esterase;Sigma) confirmed that the cell population contained z1%monocytes (36).mAbs. 4B10 (IgGl) is an anti-CD28 mAb that immunopre-

cipitates a 44-kDa disulfide-bonded dimer and enhancesproliferation and lymphokine synthesis of suboptimally ac-tivated T cells (data not shown). Indirect immunofluores-cence of CD28-transfected COS cells revealed :5% positivecells with similar intensities of staining using 4B10 or theanti-CD28 mAbs YTH 913.12 and 9.3 (8). YTH 913.12 waskindly provided by H. Waldmann (Cambridge, U.K.). Opti-mal stimulation with anti-CD28 mAb was obtained at aconcentration of 1 Ag/ml and this dose was used throughoutthe experiments. A hybridoma secreting anti-CD3 mAbOKT3 (IgG2a) was obtained from the American Type CultureCollection and the purified mAb was adhered to plastic platesat a concentration of 1 ,ug/ml. This concentration was foundto produce optimal stimulation in association with a secondsignal of T-cell activation. 4B10 and OKT3 were purifiedusing a protein A-agarose column (Bio-Rad) as described(37). The anti-B7 mAb 133 (IgM) was characterized in ourlaboratory (31, 32), and was used as ascites at a final dilutionof 1:100.B7 Transfection. The B7 cDNA clone in the pCDM8 vector

was digested with restriction endonucleases Dra I and Bgl II,and the fragment comprising nucleotides 86-1213, containingthe coding region of B7, was isolated (31). The Dra I-Bgl IIfragment was ligated into BamHI-digested, phosphatase-treated pLEN by a combination of sticky-end ligation, Kle-now polymerase fill-in, and blunt-end ligation. pLEN is aeukaryotic expression vector containing the human metal-lothionein IIA promoter, the simian virus 40 enhancer, andthe human growth hormone 3' untranslated region and poly-adenylylation site (38). pLEN was kindly provided by Met-abolic Biosystem (Mountain View, CA). Fifty micrograms ofPvu I-linearized B7-pLEN construct was cotransfected with5 ug of Pvu I-linearized SV2-Neo-Sp65 (44) into CHO-K1Chinese hamster ovary cells by electroporation using theBRL electroporator at settings of 250 V and 1600 mF.Transfectants were selected by growth in medium containingthe neomycin analogue G418 sulfate (400 jig/ml) and werecloned. Clones expressing cell surface B7, as assayed byindirect immunofluorescence with anti-B7 mAb, were re-cloned. These cells are referred to as CHO-B7 cells through-out this paper. Mock-transfected CHO-K1 (CHO-mock) cellswere made by transfection ofPvu I-linearized SV2-Neo-Sp65alone.

Cell Fixation. CHO cells were detached from tissue cultureplates by incubation in Dulbecco's phosphate-buffered salinewithout Ca2+ and Mg2' (PBS) with 0.5 mM EDTA for 30 min.Cells were washed once in PBS and resuspended in PBS at1 per ml. An equal volume of freshly prepared 0.8%paraformaldehyde in PBS was added and the cells weregently mixed for 5 min at room temperature. An equal volumeof 0.2 M lysine in PBS was added to block unreactedparaformaldehyde and the cells were pelleted by centrifuga-tion. The cells were washed once in PBS, once in RPMI 1640(Whittaker Bioproducts) containing 10% heat-inactivated fe-tal bovine serum (Sigma), resuspended in the same medium,and incubated for 1 hr in a humidified 37°C incubator. Cellswere pelleted, washed in RPMI 1640 containing 10% heat-inactivated human AB serum (North American Biologicals,Miami), 2 mM glutamine, 1 mM sodium pyruvate, penicillin(100 units/ml), streptomycin sulfate (100 Ag/ml), and gen-tamicin sulfate (5 tug/ml) (GIBCO). Cells were resuspendedin this medium containing heat-inactivated human AB serum

and 2 x 104 fixed cells were added to the appropriate wellsin a 96-well flat-bottomed microtiter plate (Nunclon; Nunc).

Proliferation Assay. CD28' T lymphocytes were incubatedin RPMI 1640 containing 10%o heat-inactivated human ABserum, 2 mM glutamine, 1 mM sodium pyruvate, penicillin(100 units/ml), streptomycin sulfate (100 pug/ml), and gen-tamicin sulfate (5 jig/ml). Cells were cultured at a concen-tration of 5 x 104 cells per 200 dul of medium in triplicatesamples in a 96-well flat-bottomed microtiter plate at 370C for3 days in 5% CO2. Cells were cultured in medium and with theappropriate stimuli added. Cells were stimulated with PMA(Calbiochem) at 1 ng/ml and ionomycin (Sigma) at 100 ng/ml(14, 15, 18). The anti-CD3 mAb was added at 1 ,tg/ml to the96-well flat-bottomed microtiter plates and incubated at roomtemperature for 1 hr; the plates were then washed twice withPBS before addition of the cells (3, 14, 39, 40). The anti-CD28mAb 4B10 was added at 1 pug/ml. The fixed CHO-B7 andCHO-mock transfectants were added at 2 x 10" cells per well.Preliminary experiments showed that maximal stimulationplateaued with the addition of 2 x 104 CHO-B7 cells. Thespecificity of the stimulation with CHO-B7 cells was assayedby the addition ofanti-B7 mAb to the cultures at a final ascitesdilution of 1:100. Over the wide range of concentrations (1:50to 1:2000) assayed, this dose was found to produce completeblocking of CHO-B7 stimulation.Thymidine Incorporation Assay. Thymidine incorporation

was used as an index of mitogenic activity. During the last 8hr ofthe 72-hour culture, the cells were incubated with 1 ,uCi(37 kBq) of [methyl-3H]thymidine (ICN Flow, Costa Mesa,CA). The cells were harvested onto filters and the radioac-tivity on the dried filters was measured in a Packard Tri-Carbscintillation counter.Lymphokine Assay. Culture supernatants were collected 24

hr after the initiation of the culture and IL-2 and IL4concentrations were assayed in duplicate using an ELISA kitaccording to the manufacturer's instructions (Quantikine; R& D Systems, Minneapolis).

RESULTSB7-Transfected CHO Cells Stimulate Proliferation of Sub-

optimally Activated CD28+ T Cells. Crosslinking of CD28 onT cells by anti-CD28 mAb has been shown to stimulate T-cellproliferation and lymphokine synthesis (18). Since B7 is anatural adhesion ligand for CD28 (30), we attempted todetermine whether binding of B7 to CD28 would deliver acostimulatory signal to T cells. To this end, a CHO cell lineexpressing high levels of B7 (CHO-B7) was constructed bystable transfection of the B7 gene under the control of thestrong metallothionein promoter. The CHO-B7 cells werefixed with paraformaldehyde and used to stimulate CD28+cells that had been suboptimally stimulated with PMA oranti-CD3.As seen in Table 1, PMA (1 ng/ml) induced a 3- to 8-fold

increase in T-cell proliferation over the medium-only con-trols. Addition of paraformaldehyde-fixed CHO-B7 cells toPMA-activated CD28+ T cells stimulated proliferation 17- to40-fold. Addition of anti-CD28 mAb also enhanced CD28+T-cell proliferation 26- to 58-fold compared with cells cul-tured with PMA alone. The stimulation by CHO-B7 was-28% less than that observed with anti-CD28 mAb in allexperiments performed. CHO-mock cells did not induceproliferation over background, providing evidence that B7was specifically inducing the proliferative signal. Neitheranti-CD28 mAb (4, 6, 8, 13) nor CHO-B7 cells were able toinduce proliferation in the untreated CD28' T cells. Culturesof the paraformaldehyde-fixed transfected CHO cells aloneshowed no proliferation over medium controls. Table 1depicts results with T cells from three representative normal

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Table 1. Effect of phorbol ester, anti-CD28, and CHO-B7 cells on proliferation of CD28+ T cells[3H]Thymidine incorporation, cpm (mean ± SEM)

CD28' cell treatment Donor 1 Donor 2 Donor 3Medium control 156 ± 65 130 ± 11 151 ± 29PMA 1,404 ± 386 1,032 ± 176 537 ± 73Anti-CD28 88 ± 18 109 + 6 188 ± 6CHO-B7 104 ± 6 102 9 143 ± 37PMA + CHO-B7 57,030 ± 1,017 34,560 ± 4,961 9,440 ± 1,103PMA + anti-CD28 82,263 ± 1,137 45,023 ± 2,684 14,215 ± 1,682PMA + CHO-mock 1,010 ± 228 728 ± 163 369 ± 36PMA + CHO-B7 + anti-B7 1,041 ± 434 737 ± 78 559 ± 52PMA + anti-CD28 + anti-B7 76,697 ± 1,241 48,776 ± 712 15,290 ± 2,011

donors, and similar results have been consistently observedin seven independent experiments.To confirm that the increased proliferation observed in the

PMA-treated CD28+ T cells was specifically mediatedthrough ligation to B7, anti-B7 mAb was added to the culturesystem to block this binding. The addition of anti-B7 mAbtotally abrogated the proliferative response induced by theCHO-B7 cells (Table 1). In contrast, anti-B7 mAb had noeffect on the stimulation of proliferation induced by anti-CD28 mAb. These results further confirm that B7 providesthe costimulatory signal.To determine whether binding of B7 to CD28 could aug-

ment proliferation ofT cells that had received a first signal ofT-cell activation through the TCR, CD28+ T cells were firstsubmitogenically stimulated with anti-CD3 mAb fixed toplastic (3, 14). Activation via the TCR provides a morephysiologic model, since the cellular events followingcrosslinking of TCR by anti-CD3 mimic the transmembranesignaling that occurs following stimulation with antigen inassociation with MHC proteins. The results obtained usinganti-CD3 stimulation are shown in Table 2 for the samenormal donors depicted in Table 1. Activation with anti-CD3mAb fixed to plastic resulted in a small, 2- to 3-fold prolif-erative response above medium controls for the majority ofdonors examined. In contrast, donor 1 demonstrated a 10-fold stimulation. This increased proliferation was presumablydue to the greater number of contaminating monocytes foundin the preparation from this donor, as the presence ofmonocytes greatly increases the stimulatory potential offixed anti-CD3 mAb (36). When CHO-B7 cells were added toanti-CD3-activated T cells, a marked increase in stimulationindex, ranging from 23- to 180-fold, was observed. Theaddition of anti-CD28 mAb also led to a marked increase inproliferation, with a stimulation index ranging from 30- to75-fold over that observed with anti-CD3 mAb alone. Thisstimulation appeared to be B7-specific, since CHO-mockcells did not augment proliferation. Addition of anti-B7 mAbcompletely blocked the proliferative response obtained withthe CHO-B7 cells but again had no effect on the responsesseen with anti-CD28 mAb. This further confirms that thestimulation occurred via binding of B7.

B7-Transfected CHO Cells Induce IL-2 but Not IL-4 Secre-tion. Stimulation of submitogenically activated T cells withanti-CD28 mAb has been shown to result in increased IL-2production (27). To determine whether-IL-2 secretion couldbe induced by ligation of B7, the culture supernatants fromcells cocultured with eitherPMA or anti-CD3 in the presenceofCHO-B7 cells or anti-CD28 were collected from the aboveexperiments and assayed for lymphokine production. Resultsof two representative donors from seven tested are depictedin Table 3. No IL-2 or IL4 was detected when the CD28+ Tcells were stimulated with PMA alone. When anti-CD28 mAbwas added to PMA-stimulated CD28+ T cells, IL-2 secretionwas markedly increased. In contrast, there was no significantincrease in IL-4 secretion over background, although positivecontrols demonstrated sensitivity and specificity of the as-say. The addition of CHO-B7 cells similarly resulted in amarked increase in IL-2 secretion but to a lesser extent,-45% of that observed with anti-CD28 mAb. As was ob-served with anti-CD28 mAb, there was no increase in IL-4production. There was no IL-2 production when resting Tcells were cocultured with either anti-CD28 mAb or CHO-B7cells (data not shown). CHO-mock cells did not induce IL-2secretion by PMA-stimulated CD28+ cells. The addition ofanti-B7 mAb specifically and nearly completely blocked thestimulation of IL-2 production by CHO-B7 cells. The anti-B7mAb had no effect on IL-2 production in the activated cellsstimulated with anti-CD28 mAb.Previous studies have demonstrated that the addition of

PMA and the calcium ionophore ionomycin strongly stimu-lates proliferation ofCD28+ cells (18). Costimulation ofCD28cells with both PMA and ionomycin enhanced proliferationby up to 75-fold compared with PMA alone in seven inde-pendent experiments. When anti-CD28 mAb or CHO-B7cells were added to PMA- and ionomycin-stimulated CD28cells, proliferation was minimally augmented, between 1- and2-fold (data not shown). However, to determine whether B7ligation could further enhance IL-2 production, PMA- andionomycin-stimulated CD28 cells were cocultured withCHO-B7 cells or anti-CD28 mAb. As seen in Table 3, CD28+cells cultured with PMA and ionomycin secreted low levelsof IL-2 and very low levels of IL-4. Addition of anti-CD28

Table 2. Effect of anti-CD3, anti-CD28, and CHO-B7 cells on proliferation of CD28' T cells[3HjThymidine incorporation, cpm (mean ± SEM)

CD28' cell treatment Donor 1 Donor 2 Donor 3Medium control 156 ± 65 130 ± 11 151 ± 29Anti-CD3 1,953 ± 631 245 ± 14 347 ± 169Anti-CD28 88 ± 18 109 ± 6 188 ± 6CHO-B7 104 ± 6 102 ± 9 143 ± 37Anti-CD3 + CHO-B7 46,543 ± 11,010 45,146 ± 4,391 35,106 ± 2,847Anti-CD3 + anti-CD28 56,836 ± 10,440 18,383 ± 5,334 26,873 ± 7,833Anti-CD3 + CHO-mock 1,618 ± 158 519 ± 135 282 ± 7Anti-CD3 + CHO-B7 + anti-B7 174 ± 5 2,377 ± 1,072 321 ± 57Anti-CD3 + anti-CD28 + anti-B7 54,646 ± 3,932 24,290 ± 14,630 30,326 ± 13,853

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Table 3. Effect of B7 on IL-2 and IL-4 production in phorbolester-stimulated CD28+ T cells

Production, pg/mlDonor 1 Donor 2

CD28+ cell treatment IL-2 IL-4 IL-2 IL-4Medium control

Proc. Natl. Acad. Sci. USA 88 (1991) 6579

1. Schwartz, R. H. (1990) Science 248, 1349-1356.2. June, C. H., Ledbetter, J. A., Gillespie, M. M., Lindsten, T. &

Thompson, C. B. (1987) Mol. Cell. Biol. 7, 4472-4481.3. Weiss, A., Manger, B. & Imboden, J. (1986) J. Immunol. 137,

819-825.4. Martin, P. J., Ledbetter, J. A., Morishita, Y., June, C. H.,

Beatty, P. G. & Hansen, J. A. (1986) J. Immunol. 136, 3282-3287.

5. Ramsdell, F. & Fowlkes, B. J. (1990) Science 248, 1342-1348.6. Weaver, C. T. & Unanue, E. R. (1990) Immunol. Today 11,

49-55.7. June, C. H., Ledbetter, J. A., Linsley, P. S. & Thompson,

C. B. (1990) Immunol. Today 58, 271-276.8. Hara, T., Fu, S. M. & Hansen, J. A. (1985) J. Exp. Med. 161,

1513-1524.9. Aruffo, A. & Seed, B. (1987) Proc. NatI. Acad. Sci. USA 84,

8573-8577.10. Turka, L. A., Ledbetter, J. A., Lee, K., June, C. H. & Thomp-

son, C. B. (1990) J. Immunol. 144, 1646-1653.11. Hansen, J. A., Martin, P. J. & Nowinski, R. C. (1980) Immu-

nogenetics 10, 247-260.12. Damle, N. K., Mohaghepour, N., Hansen, J. A. & Engelman,

E. G. (1983) J. Immunol. 131, 2296-2299.13. Ledbetter, J. A., Martin, P. J., Spooner, C. E., Wofsy, D.,

Tsu, T. T., Beatty, P. G. & Gladstone, P. (1985) J. Immunol.135, 2331-2336.

14. Manger, B., Weiss, A., Imboden, J., Laing, T. & Stobo, J. D.(1987) J. Immunol. 139, 2755-2760.

15. Wiskocil, R., Weiss, A., Imboden, J., Kamin-Lewis, R. &Stobo, J. (1985) J. Immunol. 134, 1599-1603.

16. Damle, N. K., Doyle, L. V., Grosmaire, L. S. & Ledbetter,J. A. (1988) J. Immunol. 140, 1753-1761.

17. Gmunder, H. & Lesslauer, W. (1984) Eur. J. Biochem. 142,153-160.

18. June, C. H., Ledbetter, J. A., Lindstein, T. & Thompson,C. B. (1989) J. Immunol. 143, 153-161.

19. Marlin, S. D. & Pringer, T. A. (1987) Cell 51, 813-819.20. Van Seventer, G. A., Shimizu, Y., Horgan, K. J. & Shaw, S.

(1990) J. Immunol. 144, 4579-4586.21. van Noesel, C., Miedema, F., Brouwer, M., de Rie, M. A.,

Aarden, L. A. & van Lier, R. A. W. (1988) Nature (London)333, 850-851.

22. Dustin, M. L. & Springer, T. A. (1989) Nature (London) 341,619-624.

23. Springer, T. A. (1990) Nature (London) 346, 425-434.24. Meuer, S., Hussey, R., Fabbi, M., Fox, D., Acuto, O.,

Fitzgerald, K., Hodgdon, J., Protentis, J., Schlossman, S. &Reinherz, E. (1984) Cell 36, 897-906.

25. Dustin, M. I., Olive, D. & Springer, T. A. (1989) J. Exp. Med.169, 503-517.

26. Bierer, B. E., Peterson, A., Barbosa, J., Seed, B. & Burakoff,S. J. (1988) Proc. Natl. Acad. Sci. USA 85, 1194-1198.

27. Thompson, C. B., Lindsten, T., Ledbetter, J. A., Kunkel,S. L., Young, H. A., Emerson, S. G., Leiden, J. M. & June,C. H. (1989) Proc. NatI. Acad. Sci. USA 86, 1333-1337.

28. Lindsten, T., June, C. H., Ledbetter, J. A., Stella, G. &Thompson, C. B. (1989) Science 244, 339-343.

29. Fraser, J. D., Irving, B. A., Crabtree, G. R. & Weiss, A. (1991)Science 251, 313-316.

30. Linsley, P. S., Clark, E. A. & Ledbetter, J. A. (1990) Proc.NatI. Acad. Sci. USA 87, 5031-5035.

31. Freeman, G. J., Freedman, A. S., Segil, J. M., Lee, G., Whit-man, J. F. & Nadler, L. M. (1989) J. Immunol. 143, 2714-2722.

32. Freedman, A. S., Freeman, G., Horowitz, J. C., Daley, J. &Nadler, L. M. (1987) J. Immunol. 139, 3260-3267.

33. Kawakami, K., Yamamoto, Y., Kakimoto, K. & Onoue, K.(1989) J. Immunol. 142, 1818-1825.

34. Freeman, G. J., Freedman, A. S., Rhynhart, K. & Nadler,L. M. (1990) Blood Suppl. 1, 76, 206.

35. Yamada, H., Martin, P. J., Bean, M. A., Braun, M. P., Beatty,P. G., Sadamoto, K. & Hansen, J. A. (1985) Eur. J. Immunol.15, 1164-1172.

36. Jenkins, M. K., Ashwell, J. D. & Schwartz, R. H. (1988) J.Immunol. 140, 3324-3330.

37. Van Wauwe, J. P., Demey, J. R. & Goossens, J. G. (1980) J.Immunol. 124, 2708-2712.

38. Friedman, J. S., Cofer, C. L., Anderson, C. L., Kushner,J. A., Gray, P. P., Chapman, G. E., Stuart, M. C., Lazarus,L., Shine, J. & Kushner, P. J. (1989) BiolTechnology 7,359-362.

39. Matsuyama, T., Yamada, A., Kay, J., Yamada, K. M., Ak-iyama, S. K., Schlossman, S. F. & Morimoto, C. (1989) J. Exp.Med. 170, 1133-1148.

40. Geppert, T. D. & Lipsky, P. E. (1987) J. Immunol. 138, 1660-1666.

41. Williams, I. R. & Unanue, E. R. (1990) J. Immunol. 145, 85-93.42. Mueller, D. L., Jenkins, M. K. & Schwartz, R. H. (1989) J.

Immunol. 142, 2617-2628.43. Kohno, K., Shibata, Y., Matsuo, Y. & Minowada, J. (1990)

Cell. Immunol. 131, 1-10.44. Streuli, M. & Saito, H. (1989) EMBO J. 8, 787-7%.

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