Proc. Natl. Acad. Sci. USAVol. 88, pp. 6403-6407, August 1991Immunology

Vascular cell adhesion molecule 1 induces T-cell antigenreceptor-dependent activation of CD4' T lymphocytes

(costimulation)

NITIN K. DAMLE AND ALEJANDRO ARUFFOOncogen Division, Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, WA 98121

Communicated by George J. Todaro, April 15, 1991

ABSTRACT Effective stimulation of CD4' T cells in animmune response depends on activation signals transduced vianot only the CD3-T-cell receptor (TCR) complex but also thosegenerated by accessory cell-surface proteins, including somethat mediate adhesion between T cells and antigen-presentingcells (APC). Three members of the Ig superfamily, CD54[intercellular cell adhesion molecule 1 (ICAM-1)], CD58 [lym-phocyte function-associated antigen 3 (LFA-3)], and B7, ex-pressed on the surface of APC, have been shown to mediateboth adhesion and signaling during T cell-APC interactions.Recently another member of the Ig superfamily, [vascular celladhesion molecule 1 (VCAM-1; INCAMilO)], has been iden-tified. VCAM-1 mediates adhesion between endothelial cellsand activated lymphocytes and certain tumor cells. Here, usinga soluble VCAM-1 fusion protein with receptor globulin (Rg),we examined the role of VCAM-1 in T-cell activation. Weobserved that CD4' T cells, which are inefficiently stimulatedby immobilized anti-TCR-1 or anti-CD3 monoclonal antibody(mAb) alone, can be induced to proliferate when exposed toimmobilized VCAM-1-Rg in conjunction with either immobi-lized anti-TCR-1 or immobilized anti-CD3 mAb. The costim-ulatory effects of VCAM-1-Rg on CD4' T cells is inhibited bymAb to either the CD29 (integrin 131)-CD49d [very late acti-vation antigen 4a (VLA-4a)] complex on the surface of CD4'T cells or to VCAM-1. Stimulation of CD4' T cells withimmobilized VCAM-1-Rg and anti-TCR or -CD3 mAb resultsin the synthesis of both interleukin 2 (IL-2) receptors and IL-2.In addition, anti-CD25 (anti-IL-2 receptor a) mAb significantlyinhibited the VCAM-1-Rg/anti-TCR or -CD3 mAb-drivenactivation of CD4' T cells, indicating that endogenously pro-duced IL-2 is in part responsible for the observed T-cellproliferation. Collectively, these results suggest that VCAM-1can play an important costimulatory role during the activationof CD4' T cells.

Human T lymphocytes make use of the CD3-T-cell receptor(TCR) complex during the specific recognition of antigen inthe context of products of self-major histocompatibility com-plex (MHC) genes expressed on the surface of antigen-presenting cells (APC). In addition, the CD3-TCR complexalso participates in signal transduction to initiate the activa-tion of T cells (1, 2). The interaction between the CD3-TCRand antigen-MHC product on the APC, although essential toinitiate T-cell activation, is usually not sufficient to completethe expression of T-cell functional programs. Participation ofadditional cell-surface molecules that mediate adhesionand/or signal transduction is required for optimal expressionof T-cell functions (1-8).The costimulatory functions of at least three members of Ig

superfamily expressed on the surface of APC, namely CD54[intracellular cell adhesion molecule 1 (ICAM-1)], CD58

[lymphocyte function-associated antigen 3 (LFA-3)], and B7,have been established (2, 4, 5, 7, 8). These molecules exerttheir costimulatory function by interacting with the T-cellsurface proteins CD11a/CD18 (LFA-1/P32 integrin), CD2(LFA-2), and CD28, respectively (2, 4, 5, 7, 8). In addition,resting human CD4' T cells also express on their surface, atleast three f31 (CD29) integrins that serve as receptors forextracellular matrix components such as fibronectin andlaminin to facilitate efficient cell-cell interactions. They areCD49d [very late activation antigen 4a (VLA-4a)], CD49e(VLA-5a), and CD49f (VLA-6a) (9, 10). Interaction ofVLA-4 and VLA-5 with fibronectin can also exert a costim-ulatory function in conjunction with signals transduced viathe TCR-CD3 complex (9-13).

Recently, another member of the Ig superfamily, vascularcell adhesion molecule 1 (VCAM-1), has been identified (14).Its tightly regulated expression on cytokine-activated vascu-lar endothelial cells is considered to be critical for therecruitment of primed lymphocytes to the inflamed sites (14,15). Although VCAM-1 on activated endothelial cells hasbeen implicated in supporting the CD29-CD49d complex(VLA-4)-mediated adhesion of lymphoid cells (16), it isunclear whether this molecule has any direct costimulatoryrole during T-cell activation. We have created a solublerecombinant VCAM-1-receptor globulin (Rg) fusion proteinto analyze the role of VCAM-1 during cellular interactionsunderlying the immune response. Our study shows that CD4'T cells can be stimulated to proliferate when exposed toimmobilized VCAM-1-Rg and either immobilized anti-TCR-1 monoclonal antibody (mAb) or immobilized anti-CD3mAb, but not by either immobilized mAb alone or immobi-lized VCAM-1-Rg alone. These observations suggest thatVCAM-1 may play a significant costimulatory role during theinitiation and maintenance of the immune response.

MATERIALS AND METHODSmAbs. Hybridomas OKT3 (anti-CD3), OKT4 (anti-CD4),

OKT8 (anti-CD8), OKM1 (anti-CD11b), 7G7/B6 (anti-CD25), L243 (anti-HLA-DR), and 63D3 (anti-monocyte)were obtained from American Type Culture Collection, andascitic fluids containing the respective mAb from thesehybridomas were generated in pristane-primed BALB/cmice. mAbs 9.6 (anti-CD2), G1-1 (anti-CD8), 60.1 (anti-CD11b), FC2 (anti-CD16), iF5 (anti-CD20), 9.3 (anti-CD28),and HB1Oa (anti-HLA-DR) were provided by J. A. Ledbetterfrom our institution. mAbs 4B4 (anti-CD29), 8F2 [anti-CD49d(-VLA-4a)], and 2H6 [anti-CD49e (-VLA-5a)] were providedby C. Morimoto (Dana-Farber). mAbs Al-A5 (anti-CD29)

Abbreviations: MHC, major histocompatibility complex; APC, an-tigen-presenting cells; CD, cluster designation; IL, interleukin; LFA,lymphocyte function-associated antigen; mAb, monoclonal anti-body; Rg, receptor globulin; TCR, T-cell antigen receptor; VCAM-1,vascular cell adhesion molecule-1; VLA, very late activation antigen;ELAM, endotbelial leukocyte adhesion molecule.

6403

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6404 Immunology: Damle and Aruffo

and B4G10 [anti-CD49d (-VLA-4a)] were provided by M. E.Hemler (Dana-Farber). mAb 4G7 (anti-CD19) was providedby E. G. Engleman (Stanford University School of Medi-cine). mAbs 2G7 and 1E7 (anti-VCAM-1) and 7A9 [anti-endothelial leukocyte adhesion molecule (anti-ELAM-1)]were provided by W. Newman (Otsuka America Pharma-ceutical, Rockville, MD). mAbs P1E6 [anti-CD49b (-VLA-2a)], P1B5 [anti-CD49c (-VLA-3a)], P4G9 [anti-CD49d(-VLA-4a)], and P1D6 [anti-CD49e (-VLA-5a)] were ob-tained from Telias Pharmaceuticals (San Diego). mAb WT-31(anti-TCR-1) was provided by W. Tax (University of Ni-jmegen, The Netherlands) and also was obtained from BectonDickinson. Anti-Tac [CD25, also called interleukin 2 (IL-2)receptor a] mAb was provided by T. A. Waldmann (NationalInstitutes of Health). Each of the above mAbs is an IgGantibody. Fluorescein isothiocyanate (FITC)-labeled or phy-coerythrin-labeled mAb directed against various lymphoidsurface molecules were obtained from AMAC or Coulter.

Construction of VCAM-1-Rg. VCAM-1 cDNA sequencesencoding the three amino-terminal Ig-like domains (14) wereamplified by the polymerase chain reaction (PCR) withsynthetic oligonucleotides complementary to sequencesflanking this region and 0.1 mg of a cDNA library preparedfrom IL-1-stimulated human umbilical vein-derived endothe-lial cells linearized with the restriction enzyme Mlu I. Typ-ically, 30 cycles were conducted consisting of 30 s at 92°C, 2min at 55°C, and 3 min at 72°C; the reaction buffer recom-mended by the enzyme vendor (United States Biochemical)was used. Oligonucleotides were designed to allow the cre-ation of restriction enzyme cleavage sites at the 5' and 3'extremities of the amplified cDNA segments to facilitatesubsequent insertion into the IgGl expression vector (17).The VCAM-1 primer, encoding sequences immediately up-stream of the signal peptide and including an Xho I site, wassynthesized with the following sequence: 5'-GCG ACT AGTCTC GAG CTT AAA ATG CCT GGG AAG ATG GTC-3'.A reverse primer containing a HincII restriction site wassynthesized with the following sequence: 5'-CAC CGT CGACTC ACC TGC TTC AAC AAT TAA TTC CAC CTC-3'.VCAM-1 PCR products were digested with Xho I and HincIIand ligated to the Xho I/Sca I-cut CD8-IgG1 vector (17). Theresulting construct was transfected into COS cells and thedesired fusion protein was recovered from the supernatant asdescribed (17).

Isolation of CD4+ T Cells. Peripheral blood mononuclearcells from healthy donors were separated by Ficoll/Hypaquedensity gradient centrifugation. Resting CD4+ T cells wereisolated by rigorous immunomagnetic negative selection us-ing Dynabeads M-450 (Dynal, Great Neck, NY) as described(9, 10). Negative selection was performed by using a mixtureof mAbs: 63D3 against human monocytes; L243 or HB1Oaagainst HLA-DR on B cells, monocytes, and activated Tcells; 4G7 against CD19 or iF5 against CD20 on B cells;OKM1 or 60.1 against CD11b on monocytes and natural killer(NK) cells; FC2 against CD16 on NK cells; and OKT8 orG10-1 against CD8 on CD8+ T cells. The purity of the isolatedCD4+ populations was >95% as assessed by direct or indirectimmunofluorescence analysis using a fluorescence-activatedcell sorter (EPICS V, Coulter). Isolated CD4+ T cells wereresuspended at 1 x 106 cells per ml in complete mediumconsisting of RPMI 1640 supplemented with 100 units ofpenicillin and 100 ,ug of streptomycin per ml, 2 mM L-gluta-mine, 50 ,AM 2-mercaptoethanol, and 5% (vol/vol) fetalbovine serum. These CD4+ T cells were unable to proliferatein response to mitogenic concentrations of phytohemagglu-tinin (10 ,ug/ml) or soluble anti-CD3-TCR complex (100ng/ml) in the absence of accessory cells.

Proliferation Assays. Round-bottom microtiter plates(Corning) were precoated overnight at 40C with a mixture (10Ag/ml each) of affinity-purified goat anti-mouse and anti-

human IgG Fc antibodies (Tago) (20 /4 per well in sodiumbicarbonate buffer, pH 9.6) after which additional protein-binding sites were blocked overnight with 2% bovine serumalbumin in RPMI 1640. VCAM-1-Rg (Rg-human IgG Fcparticle) and anti-TCR-1 (mouse IgG) mAb at various con-centrations were immobilized in the above microtiter wellsfor 2 hr, and the plates were washed twice with completemedium as described (18). These plates "armed" withVCAM-1-Rg and/or anti-TCR-1 were then used to stimulateCD4' T cells. Fifty-thousand CD4' T cells in 0.1 ml ofcomplete medium were cultured in the above armed micro-titer plates for 96 hr at 370C in a5% C02/95% air atmosphere.Proliferative responses in these cultures were measured onday 4 by pulsing triplicate cultures with 1 uCi (37 kBq) of[3H]thymidine (6.7 Ci/mmol; New England Nuclear) per well16 hr before harvesting of cells for the measurement ofradiolabel into newly synthesized DNA. The results areexpressed as cpm ± SEM.

Production of IL-2 and Analysis of IL-2 Receptor Expres-sion. Fifty-thousand resting CD4' T cells were cultured in96-well microtiter plates previously armed with VCAM-1-Rg(50 ng per well), anti-TCR-1 (50 ng/ml), or both as describedabove. Cell-free supernatants from these cultures were col-lected after 48 hr and assayed for the IL-2 activity by usingthe IL-2-dependent T-cell line CTLL2 as described (19). TheIL-2 concentration of each sample was calculated by refer-ence to recombinant IL-2 (1.6 x 107 international units/mg;Cetus) and expressed in international units/ml.For the analysis of CD25 (IL-2 receptor a)' CD4+ T cells,

cultures were harvested after 72 hr and stained with fluores-cein-conjugated 7G7/B6 or anti-CD25 mAb (AMAC) andanalyzed by fluorescence-activated cell sorting (EPICS C,Coulter).

RESULTS

Preparation of Soluble VCAM-l-Rg Fusion Protein. Thesoluble VCAM-1-immunoglobulin fusion protein (VCAM-1-Rg) used in this study was created by fusing a cDNA fragmentencoding the amino-terminal signal sequence and the firstthree Ig-related domains of VCAM-1 (residues 1-281) to agenomic DNA fragment encoding the constant region ofhuman IgG1 (Fig. 1A) as described (14, 17). The resultingVCAM-1-Rg was produced by appropriately transfectedCOS cells. VCAM-1-Rg was recognized by anti-VCAM-1mAbs 2G7 and 1E7 and, upon immobilization to plastic,supported adherence of activated CD4+ T cells (ref. 20;N.K.D., unpublished data).A CD7-Rg fusion protein to be used as a control for

nonspecific, Fc-mediated interactions was prepared by fus-ing a cDNA fragment encoding the extracellular domain ofCD7-Rg to a genomic DNA fragment encoding the constantregion of human IgG1 (A.A. and B. Seed, unpublished data).Both VCAM-1-Rg (molecular mass, -75 kDa under reducingconditions and -160 kDa under nonreducing conditions) andCD7-Rg (-55 kDa under reducing conditions and -120 kDaunder nonreducing conditions) are disulfide-linked dimers(Fig. 1B) and accumulate in the supernatant of transfectedCOS cells to a concentration of -100 ng/ml 4 days post-transfection.

Costimulatory Effects of VCAM-l-Rg. The ability ofVCAM-1 to provide costimulatory signals to CD4+ T cellsactivated via the CD3-TCR complex was examined. BothVCAM-1-Rg and anti-TCR-1 mAb were immobilized in mi-crotiter wells precoated with a mixture of affinity-purifiedgoat antibodies recognizing human and mouse IgG Fc asdescribed. Freshly isolated, resting CD4' T cells were addedto the microtiter wells, and their proliferation was monitoredby the incorporation of [3H]thymidine. CD4' T cells in wellscontaining immobilized VCAM-1-Rg and anti-TCR-1 mAb

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

Proc. Natl. Acad. Sci. USA 88 (1991) 6405

Ss

A Ig-r Ig-r .Ig-r H CH. H3 VCAM-1 RgEVELIV NAEPK

B1 2

-200

-97

-69

-46

FIG. 1. VCAM-1-Rg fusion gene and protein. (A) Antibodyexons (human IgGl) are stippled, and introns are represented byconnecting lines. H, CH2, and CH3 denote the IgG hinge, CH2, andCH3 constant region exons, respectively. The VCAM-1 extracellulardomain is represented by a box. Ig-r denotes immunoglobulin-relateddomains, and SS indicates the secretory signal sequence. The aminoacid sequence (single-letter code) predicted at the site of fusion of theextracellular domain fragment of VCAM-1 (roman letters) and hu-man IgG1 (italic letters) is shown below the diagram. (B) Radiola-beled fusion proteins were purified by adsorption to and elution froma protein A-Trisacryl column as described (17) and were separated byelectrophoresis under nonreducing conditions (lane 1) or reducingconditions (lane 2). The electrophoretic mobility of molecular massstandards is shown in kDa.

showed a strong proliferative response (Fig. 2), while CD4'T cells in wells coated with either VCAM-1-Rg and anti-CD19 mAb (reactive with CD19 expressed on the surface ofB cells) or CD7-Rg and anti-TCR-1 mAb failed to proliferate.Proliferation of CD4' T cells required the presence of bothimmobilized VCAM-1-Rg and immobilized anti-TCR-1 mAb.CD4' T cells in wells containing either soluble VCAM-1-Rgand immobilized anti-TCR-1 mAb or immobilized VCAM-1-Rg and soluble anti-TCR-1 mAb did not proliferate (Fig. 2).

In solution Anti-CD1 9

Immobilized Anti-CD1 9

In solution Anti-TCR-1

Immobilized Anti-TCR-1

In solution CD7 Rg

Immobilized CD7 Rg

In solution VCAM-1 Rg

Immobilized VCAM-1 Rg

A ImmobilizedO CD7 Rg

R VCGM-1 Rg

0 10 20 30 40 50

ImmobilizedB [ Anti-CD1 9

M Anti-TCR-1

. . -. . I0 10 20 30

3H[dThd] incorporation,cpm x 10-3 + SEM

40

FIG. 2. Ability ofsoluble and immobilized forms ofVCAM-1-Rg,CD7-Rg, anti-TCR-1 mAb, or anti-CD19 mAb to costimulate pro-liferation of CD4+ T cells. Fifty thousand resting CD4+ T cells werecultured with immobilized VCAM-1-Rg or CD7-Rg (50 ng per well)in the presence of soluble (100 ng/ml) or immobilized (100 ng perwell) forms of either anti-TCR-1 or anti-CD19 mAb (A) or withimmobilized anti-TCR-1 or anti-CD19 mAb (100 ng per well) in thepresence of soluble (50 ng/ml) or immobilized (50 ng per well) ofVCAM-1-Rg or CD7-Rg in a final volume of 0.1 ml of completemedium per microtiter well (B). Cellular proliferation in thesecultures was measured on day 4.

We examined the concentration-dependance of the immo-bilized VCAM-1-Rg/anti-TCR-1 mAb activation of CD4' Tcells. CD4' T cells were incubated in wells that had beencoated with either various concentrations of VCAM-1-Rg(0.05-50 ng per well) and a fixed concentration of anti-TCR-1mAb (100 ng per well) or various concentrations of anti-TCR-1 mAb (0.1-100 ng per well) and a fixed concentrationof VCAM-1-Rg (50 ng per well), and their proliferativeresponse was measured. CD4' T cells in wells containing afixed amount of immobilized anti-TCR-1 mAb showed in-creasing proliferation with increasing quantities of immobi-lized VCAM-1-Rg (Fig. 3 Upper). Likewise, CD4' T cells inwells containing a fixed quantity ofimmobilized VCAM-1-Rgexhibited proliferative responses that were proportional tothe concentration of immobilized anti-TCR-1 mAb (Fig. 3Lower). In parallel experiments, CD4' T cells incubated inwells containing various amounts of immobilized CD7-Rgwith a fixed concentration of immobilized anti-TCR-1 mAb(Fig. 3 Upper) or various amounts ofimmobilized anti-TCR-1mAb with a fixed concentration ofimmobilized CD7-Rg (Fig.3 Lower) failed to proliferate.The ability of mAb directed at CD2, CD3-TCR, CD19, or

CD28 molecules coimmobilized with VCAM-1-Rg orCD7-Rg to stimulate proliferation of CD4' T cells was alsoexamined. CD7-Rg or VCAM-1-Rg (50 ng per well) werecoimmobilized with anti-CD2, anti-CD3, anti-TCR-1, anti-CD19, or anti-CD28 (each at 100 ng per well) in microtiterwells, and the proliferative response ofCD4' T cells in thesewells was measured after 96 hr. The costimulatory effect ofVCAM-1-Rg on the proliferation of CD4' T cells was ob-served only when these cells were also costimulated withimmobilized mAb directed at the CD3-TCR complex (anti-CD3 or anti-TCR-1) but not CD2 or CD28 molecules (data notshown). These results suggest that costimulatory effect ofVCAM-1-Rg is dependent on the activation signals receivedvia ligation of the CD3-TCR complex and that similar ligationof CD2 or CD28 on the surface of resting T cells is notsufficient to enable these T cells to be costimulated by

wLL

+1CO

0

xE0.-

01600.0

*0

Q

F._-

30Immobilized Anti-TCR-1 +

24 *-O VCAM-1 Rg18 -O-CD7Rg

12

6

0.01 .1 1 10 100Immobilized fusion protein, ng per well

30Immobilized

* VCAM-1 Rg18 - CD7Rg

+ Anti-TCR-112

6

0.11m 1.1 1I.01 .1 1 1 10 10ll.01 .1 1 10 100 1000

Immobilized anti-TCR-1, ng per well

FIG. 3. Concentration dependence of the costimulatory effect ofimmobilized VCAM-1-Rg and anti-TCR-1 mAb. (Upper) VCAM-1-Rg or CD7-Rg at various concentrations was immobilized withanti-TCR-1 mAb (100 ng per well). (Lower) Similarly, variousconcentrations of anti-TCR-1 were immobilized with either VCAM-1-Rg or CD7-Rg (50 ng per well). Fifty thousand CD4+ T cells in 0.1ml of complete medium were added to each microtiter well, and theirproliferative response was examined after 96 hr.

Immunology: Damle and Aruffo

6406 Immunology: Damle and Aruffo

VCAM-1-Rg. These results further underscore the impor-tance of the signals received via the CD3-TCR complex onthe surface of T cells in inducing responsiveness to othercostimuli such as VCAM-1.

Costimulatory Effect ofVCAM-1-Rg Involves the IL-2/IL-2Receptor System. The ability of immobilized VCAM-1-Rg/anti-TCR-1 mAb to induce the expression of IL-2 receptorsand IL-2 by CD4+ T cells was examined. CD4+ T cells werestimulated with immobilized anti-TCR-1 mAb, immobilizedVCAM-1-Rg, or both for 72 hr. After stimulation the levelsof cell-surface CD25 (IL-2 receptor a) were monitored byflow cytometry. Binding of CD4+ T cells to the immobilizedVCAM-1-Rg/anti-TCR-1 mAb induced the expression ofCD25 on CD4+ T cells to significantly higher levels (>60%oCD25+) than-those observed on cells incubated with eitherimmobilized VCAM-1-Rg (<10o CD25+) or immobilizedanti-TCR-1 mAb (<20% CD25+) (Fig. 4). Similar results wereobserved in IL-2 production assays. CD4+ T cells incubatedin wells coated with VCAM-1-Rg and anti-TCR-1 mAbproduced significantly more IL-2 (2.5-6 units/ml) than cellsthat had been incubated in wells coated with either VCAM-1-Rg or anti-TCR-1 mAb alone (<0.5 unit/ml). In addition,anti-CD25 mAb (anti-IL-2 receptor a) significantly inhibitedthe proliferation of CD4+ T cells elicited by immobilizedVCAM-1-Rg/anti-TCR-1 mAb (Table 1).Role of VLA-4 Integrin During Costimulation with

VCAM-1. Human resting CD4+ T cells express at least threedistinct VLA-a (integrin) molecules in association with acommon /31 (CD29) subunit: VLA-4a (CD49d), VLA-5a(CD49e), and VLA-6a (CD49f) (9, 10). Recently, the CD49d-CD29 complex, VLA-4, has been identified as the majorreceptor on lymphoid cells for VCAM-1 (16). To study therole of 31 integrins in the VCAM-1/TCR-mediated activationof CD4+ T-cells, VCAM-1-Rg and anti-TCR-1 mAb wereimmobilized in microtiter wells. Subsequently, CD4+ T cellswere added to the wells in conjunction with different anti-CD29 (VLA-f) mAb or anti-VLA a-chain mAb (10 ,ug/ml).Anti-CD29 mAb 4B4 or anti-CD49d (VLA-4a) mAb 8F2caused almost complete inhibition of the VCAM-1-Rg/anti-TCR-1 mAb-mediated activation ofthe CD4+ T cells (Fig. 5).Two other anti-CD49d mAbs (B5G1O and P4G9) were alsoinhibitory, although less effective than mAb 8F2, perhapsowing to their reactivity with distinct epitopes on the CD49d(VLA-4a) molecule (6, 9-13). Another anti-CD29 mAbAl-A5 was ineffective in inhibiting VCAM-1-Rg/anti-TCR-1mAb-induced stimulation of T cells. Partial but consistentinhibition was observed with one anti-CD49e (VLA-5a)mAb, 2H6. The modulatory effect of 2H6 also appears to beepitope-dependent since another anti-CD49e (VLA-5a)mAb, P1D6 had no inhibitory effect in this assay. Two otheranti-a chain mAb [anti-CD49b (VLA-2a), P1E6; anti-CD49c(VLA-3a), PlB5] were also ineffective in modulating thecostimulatory effects mediated by immobilized VCAM-1-Rgand anti-TCR-1 mAb.

In.0Ez

c.

.VCAM-1Rg---Anti-TCRl

A - Anti-TCRI+VCAM-1 Rg

LogIFluorescence"Intensity_Log Fluorescence Intensity--

FIG. 4. Expression ofIL-2 receptor on CD41 T cells derived fromcultures stimulated with immobilized VCAM-1-Rg and anti-TCR-1mAb. CD4+ T cells were cultured for 72 hr with either immobilizedVCAM-1-Rg (50 ng per well) or immobilized anti-TCR-1 (50 ng perwell), or both, after which their surface expression ofCD25 moleculewas examined by direct immunofluorescence analysis.

Table 1. Anti-Tac mAb inhibits CD4' T-cell proliferationinduced by immobilized anti-TCR-1 mAb and-VCAM-1-Rg

[3H]dThd incorporation (cpm ± SEM X 10-3)in cultures

Culture medium Addition to medium

Exp. alone 7G7 or B6 Anti-Tac1 23.2 ± 1.6 24.7 ± 1.6 10.1 ± 0.6 (59)*2 41.2 ± 3.2 40.4 ± 2.0 11.8 ± 1.4 (71)*3 35.1 ± 3.1 33.9 ± 1.8 9.6 ± 0.8 (73)*Fifty thousand CD41 T cells were cultured in microtiter wells with

immobilized VCAM-1-Rg and anti-TCR-1 mAb in the presence of 10.tg of soluble anti-CD25/IL-2 receptor a mAb 7G7 or B6 or ofanti-Tac. Proliferative response in these cultures was measured onday 4 of culture. Background uptake in the absence of immobilizedinducers was always <300 cpm.*Figures in parentheses denote % inhibition of the response.

In parallel experiments, the ability ofanti-VCAM-1 mAb toblock the immobilized VCAM-1-Rg/anti-TCR-1 mAb-mediated activation of CD4' T cells was also examined.Anti-VCAM-1 mAb 2G7 but not 1E7 (each reactive withdistinct epitope on VCAM-1; ref. 20) almost completelyinhibited activation (Fig. 6A), whereas in a control experi-ment, the anti-ELAM-1 mAb 7A9 (20) showed no inhibitoryeffects. These results are consistent with earlier observationsthat the 2G7-reactive but not the 1E7-reactive epitope ofVCAM-1 is involved in interactions with T cells (20).

DISCUSSIONThis study establishes that CD4+ T cells can be induced toproliferate by interacting with coimmobilized VCAM-1-Rgand either anti-TCR-1 or anti-CD3 mAb. These observationsallow us to include VCAM-1 into the subgroup of Ig super-gene family members (14) functioning as adhesion/activationmolecules, which also includes CD54 (ICAM-1), CD58 (LFA-3), and B7. Stimulation of CD4+ T cells with VCAM-1-Rgwas absolutely dependent on the costimulation of the CD3-TCR complex and required that both VCAM-1-Rg and eitheranti-TCR-1 or anti-CD3 mAb be coimmobilized.

Blocking mAb

Anti-VCAM-1 11:7

Anti-VC4M-1 2GC7

Anti-ELAM-1 7A9

Anti-CD29 (,31) Al-A5

Anti-CD29 (,1 ? 4B4

Anti-CD49b (VLA-2w)Anti-CD49c (VLA-3a)Anti-CD49d (VLA-4a)Anti-CD49d (VLA-4a)Anti-CD49d (VLA-4a)Anti-CD49e (VLA-5a)Anti-CD49e (VLA-Sa)

PI E6P1 B58F2

B5C1 oP4G92H6

P1 D6

--I

z

B-i

,~~~~~~~~~~~~~~~..,.,.--H

_ . .. - _ . ...-i.._

0i 20(t 4 0 E J8O0 1 ((

% Inhibition

FIG. 5. Effect of mAb reactive with various 81 integrins on theproliferative response of CD4+ T cells to immobilized VCAM-1-Rgand anti-TCR-1 mAb. Fifty thousand CD4+ T cells were culturedwith immobilized VCAM-1-Rg and anti-TCR-1 mAb in a finAlvolume of 0.1 ml of complete medium. Various mAb reactive witheither a subunits or bl subunit ofVLA molecules were individuallyadded at 10 ,g/ml to the indicator cultures, and their proliferativeresponse was measured on day 4. mAbs reactive with eitherVCAM-1 or ELAM-1 were similarly examined.

Proc. NatL Acad. Sci. USA 88 (1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 6407

Stimulation of CD4' T cells with immobilized VCAM-1-Rg and anti-TCR-1 mAb-induced both the expression ofcell surface IL-2 receptors (CD25) and the secretion of IL-2.Exogenous addition of cytokines such as IL-1, IL-2, IL-4, orIL-6 did not significantly increase the proliferative response(N.K.D., unpublished data). Furthermore, anti-CD25 mAbreactive with the high-affinity functional IL-2 receptors wasable to inhibit the VCAM-1-Rg/anti-TCR-1 mAb-mediatedactivation of CD4' T cells. These two results togethersuggest that the immobilized VCAM-1-Rg/anti-TCR-1 mAb-mediated activation of CD4' T cells generates sufficientgrowth-promoting cytokines to sustain the propagation ofresponding CD4' T cells.Human resting CD4' T cells express on their surface at

least three VLA-a (integrin) molecules in association with acommon f1 (CD29) subunit: CD49d (VLA-4a), CD49e (VLA-5a), and CD49f(VLA-6a) (9, 10). The CD49d-CD29 complex(VLA-4) on lymphoid cells has been identified as a receptorfor VCAM-1, which in part is responsible for mediatingleukocyte-endothelial cell adhesion (16). Therefore, we ex-amined the role of f1 integrins on the VCAM-1/anti-TCR-driven activation of CD4' T cells. Antibody 4B4 directedtowards the CD29 (,81) molecule almost completely inhibitedVCAM-1-Rg/anti-TCR-1 mAb-mediated activation of CD4+T cells. Similarly, each of the three mAb directed at theCD49d (VLA-4a) was found to be inhibitory. In contrast,mAb directed at CD49b (VLA-2a) or CD49c (VLA-3a) hadno effect on the above response.Of interest was the effect of the anti-CD49e (VLA-5a) mAb

2H6, which consistently but partially inhibited the responseof CD4+ T cells to the immobilized VCAM-1-Rg/anti-TCR-1mAb. Recently, both CD49d (VLA-4a) and CD49e (VLA-5a)have been shown to stimulate CD4+ T-cell proliferation viabinding to immobilized fibronectin in the presence of anti-TCR-CD3 mAb. CD29-CD49e complex (VLA-5) recognizesthe Arg-Gly-Asp sequence-containing region of fibronectin,whereas CD29-CD49d complex (VLA-4) recognizes theCS-1 sequence in the IIICS region of fibronectin (9-13, 21).Efficient costimulation of T cells with fibronectin requirescooperative interactions between both the VLA-4 and VLA-5molecules (10-13). The mAb blocking results described in thepresent study (Fig. 4) suggest that similar cooperative inter-actions between VLA-4 and VLA-5 may be involved in theVCAM-1-Rg/anti-TCR-mediated activation ofCD4+ T cells.Although VLA-4 has been identified as the main receptor

for VCAM-1, the role of VLA-5 during the interaction ofVLA-4+/VLA-5+ cells with VCAM-1 has not been formallyruled out. The contribution of VLA-5 during the adhesion oflymphoid cells to VCAM-1 was not examined in the report,which established the receptor-counterreceptor relationshipbetween VLA-4 and VCAM-1 (16). The VLA-5 complex maypossess weaker avidity for VCAM-1 than VLA-4. It might beworthwhile to reexamine carefully the ability of anti-CD49e(VLA-5a) mAb to block adhesion of lymphoid cells toVCAM-1.Considering the involvement of VLA-5 during fibronectin-

mediated CD4+ T-cell stimulation (10-13), a possibility ex-isted that the VCAM-1-Rg-mediated activation reported heremay in part be attributable to the fibronectin present in the 5%fetal bovine serum used in some of the experimental steps.However, this is unlikely because under the experimentalconditions under which VCAM-1-Rg stimulated CD4+ T-cellproliferation, CD7-Rg was unable to do so. Moreover, Arg-Gly-Asp-containing and CS-1 peptides, which block thecostimulatory effect of immobilized fibronectin, do not affectthe costimulatory effect of VCAM-1-Rg (unpublished data).Furthermore, soluble fibronectin, unlike immobilized fibro-nectin, does not function as costimulatory agent (12), and its

immobilization during the culture period also seems unlikelydue to prior blocking of protein-binding sites by BSA.VCAM-1 is an inducible, transmembrane, adhesion mole-

cule expressed primarily on inflamed vascular endothelialcells (14, 15). Its expression is upregulated on endothelialcells that have been activated by the proinflammatory cy-tokines such as IL-1 or tumor necrosis factor (14, 15).Activated endothelial cells are capable of presenting antigento CD4' T cell by virtue of their expression of class II MHCmolecules (22). Thus VCAM-1' class II MHC' endothelialcells may play a crucial role in recruiting antigen-reactiveCD4' T cells to sites of chronic inflammation. RecentlyVCAM-1 expression on interdigitating follicular dendriticcells in the germinal centers of immunologically involved/activated lymphoid organs has also been reported (15, 23). Itis thought that activated T and B cell-binding to dendriticcells at these sites is in part mediated by VCAM-1-VLA-4interaction (23). These observations, in conjunction with theexperiments presented here, suggest that VCAM-1 on certainAPC may play an important role in sustaining the activationof CD4' T cells by mediating the binding to, and therebycostimulating proliferation of, antigen-responsive VLA-4' Tcells.

We thank -various investigators for generously providing mAb;Chris Eberhardt, Mary LaMountain, and Monica Van der Vieren forexpert assistance; Derek Hewgill for help with flow cytometry; Drs.Jeffrey A. Ledbetter and Peter S. Linsley for stimulating discussions;and Drs. Ingegerd Hellstrom, Karl-Erik Hellstrom, and Brian Seedfor continued support and encouragement.

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Immunology: Damle and Aruffo