Expression and distribution of CD11aICD18 human T cell-B cell interactions Shigeto

Tohma,

Jane

Simmoas

Arthritis

Harold

C.

Center,

Dallas

Abstract: Interactions molecule 1 (ICAM-1, tion-associated antigen critical role in T cell-B

E. Ramberg, Research

and

Center,

Peter

Department

between intercellular adhesion CD54) and leukocyte func1 (LFA-1, CD11aICD18) play a cell collaboration. The current cx-

periments were carried out to determine the expression and distribution of these adhesion molecules on human peripheral T cells and B cells during T cell-B cell collaboration. Resting CD4 T cells were largely ICAM-1 negative, whereas immobilized anti-CD3 monoclonal antibody (mAb) rapidly induced ICAM-1 expression. By contrast, most B cells expressed ICAM-1 before activation, and further increases in density were noted with stimulation. Both B cells and CD4 T cells expressed LFA-1 before activation, although the density on CD4 T cells was considerably greater. A double staining method for electron microscopic analysis was developed that permitted analysis of the expression and distribution of ICAM-1 to be assessed during T cell-B cell collaboration. Under the experimental conditions examined, B cells showed a uniform distribution of ICAM-1. In contrast, ICAM-1 was highly mobile on the surface of CD4 T cells. If the T cells were not fixed, staining, even at 4#{176}C,caused rapid redistribution of ICAM-1 into aggregates. However, by fixing cells before the staining procedures, the distribution of ICAM-1 on CD4 T cells could be accurately assessed. Most (85%) of the fixed activated CD4 T cells showed a uniform distribution of ICAM-!. However, when activated CD4 T cells were cocultured with B cells, redistribution of ICAM-1 on CD4 T cells but not B cells occurred, such that the majority (85 %) was found at or immediately adjacent to the point of attachment to the B cells. No redistribution of LFA-1 on either T cells or B cells was found. These findings suggest that rapid changes in density of ICAM-1 expression and the mobility of ICAM-1 on activated T cells may play a role in providing activation signals to B cells during T cell-B cell collaboration. Key

J.

Leukoc.

Words:

.

leulcocyte

.

CD54

Biol.

52:

intercellular

97-103; adhesion

function-associated .

T cell-B

cell

1992.

antigen

molecule 1(LFA-1)

1

(ICAM-1)

In the

role

E. Lipsky of Internal

Medicine,

system, in

various

University

of Texas

Southwestern

Medical

terreceptors, including CD54 (ICAM-1) [10-12], have been shown to mediate a variety ofadhesion-dependent events, including a number of those involved in the functional activities of lymphocytes. Much of the understanding of the involvement of interactions of LFA-1 and its ligands in lymphocyte function has been defined by use of monoclonal antibodies (mAbs) and includes a role in the adhesion of cytotoxic T cells and natural killer (NK) cells to target cells and the physical interactions betwten responding T cells and antigen presenting cells [1-4]. Si#{241}ilarly, LFA-1 plays a role in the development of cell-to-cell contacts required for some T cell-dependent B cell responses [5-7, 9]. ICAM-1 (CD54) is widely distributed on cells of both hematopoietic and nonhematopoietic origin [13]. It is cxpressed at low levels on peripheral blood cells and at higher levels on mitogen-activated T lymphoblasts, Epstein-Barr virus-transformed B cells, and some cell lines of T cell and myeloid lineage, whereas LFA-1 is constitutively expressed on the large majority of lymphocytes and accessory cells [10, 13, 14]. However, the kinetics of expression of these molecules after activation and the distribution of these adhesion molecules at the site of cellular interactions have not been delineated. The current studies were undertaken, therefore, to investigate in detail the expression and distribution of LFA-1 or ICAM-i molecules on human lymphocytes. The focus of these studies was the distribution of LFA-i and ICAM-i during T cell-B cell collaboration, as interactions between these adhesion molecules have been shown to play a central role in T cell-dependent polyclonal activation of B cells [9]. The current studies show that ICAM-1 expression on the surface of activated CD4 T cells is dynamic and highly mobile. Thus, B cells exhibit a uniform distribution of ICAM-1 under the experimental conditions examined. By contrast, when activated CD4 T cells were cocultured with B cells, redistribution of T cell ICAM-1 to the point of attachment to the B cell was observed. These results suggest that redistribution of ICAM-1 of CD4 T cells may play a role in facilitating T cell-B cell collaboration.

interaction

Abbreviations: fluorescence-activated

immune

during

CDJJaJCDJ8

INTRODUCTION

portant

and CD54

direct immune

cell-to-cell responses,

contact including

plays

an

im-

cytotox-

icity [1-3], antigen-presenting cell-T cell interactions [4], and T cell-B cell collaboration [5-9]. It has been shown that several glycoproteins on the cell surface mediate cell-to-cell interactions. One of these adhesion molecules, CD11a/CD18 (LFA-i), a member of the j32 integrin family, is expressed by all leukocytes. Interactions mediated by LFA-1 and its coun-

isothiocyanate;

ATCC, cell HLA,

human

American sorter; FCS,

Type fetal

leukocyte

antigen;

Culture calf serum; ICAM-l,

Collection; FI1C, intercellular

FACS, fluorescein adhe-

sion molecule 1; IgG2a, immunoglobulin G2a IL-l, interleukin-l; LFA-I, leukocyte function-associated antigen 1; mAb, monoclonal antibody; NK, natural killer; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; SA, Staphylococcus aureus; SRBC, sheep red blood cell. Reprint requests: Peter Lipsky, Harold C. Simmons Arthritis Research Center, Dept. of Internal Medicine, U. of Texas Southwestern Medical Center, Dallas, TX 75235, USA. Received January 9, 1992; accepted March 16,

Journal

of Leukocyte

Biology

Volume

52, July

1992

97

MATERIALS

AND

Monoclonal Various Culture (IgG2a) OKT3 molecular (ATCC),

METHODS

cells [24]. The neuraminidase-treated diatrizoate-Ficoll resulting population

Antibodies

mAbs were used, including OKTi1 (American Type Collection [ATCC]), an immunoglobulin G2a mAb directed at the CD2 molecule on T cells; (A’ICC) and 64.1, IgG2a mAbs directed at the CD3 complex on mature T cells [15, 16]; OKT8 an IgG2a mAb directed at the CD8 molecule; R7.1

(a gift of Dr. Robert Rothlein, Boehringer maceuticals, Ridgefield, CT), an IgGi CD11a; 60.3 (a gift of Dr. Patrick Beatty, Cancer Center, Seattle, WA), an IgG2a CD18; RFB-4 (a gift ofDr. Ellen Vitetta, Southwestern Medical Center at Dallas, IgGi mAb directed at CD22 on B cells;

Ingelheim PharmAb directed at Fred Hutchinson mAb directed at University of Texas Dallas, TX), an R6.5 (a gift of Dr.

Robert Rothlein), an IgG1 mAb directed at CD54 (ICAM-1); L243 (ATCC), an IgG2a mAb directed at monomorphic human leukocyte antigen HLA-DR determinants [17]; P1.17 (ATCC), a control IgG2a mAb.

Reagents Formalinized purchased was used

Cowen I strain Staphylococcus aureus (SA) from Calbiochem-Behring, San Diego, CA, at a concentration of 1:60,000 (v/v). Recombinant

interleukin-2 Roche, Nutley,

Preparation Studies

(IL-2) NJ.

was

of Antibodies

obtained

from

for Electron

was and

Microscopic

Fifteen-nanometer particles of colloidal gold, prepared according to Slot and Geuze [18] were conjugated to RFB-4 (an anti-CD22 mAb) by a previously described procedure [19] and R6.5 (an anti-CD54 mAb) and P1.17 (control mAb) were biotinylated as described [20]. Horseradish peroxidase-labeled streptavidin was purchased from Vector Laboratories, Burlingame, CA.

Culture

Medium

All cultures were carried ton Biologics, Lenexa, (200 U/ml), gentamicin mg/mi), and 10% fetal

out in medium KS) supplemented (10 tg/ml), bovine serum

RPMI 1640 (Hazelwith penicillin G L-glutamine (0.3 (Gibco).

Cell Preparation Peripheral

blood

mononuclear

cells

from healthy adult volunteers heparinized venous blood over gradients (Sigma Chemical Co.,

Cell

(PBMCs)

were

obtained

by centrifugation sodium diatrizoate-Ficoll St. Louis, MO) [21].

of

PBMCs were separated into T cell-enriched and B cell-enriched populations as described [22]. Briefly, PBMCs were depleted of monocytes and NK cells by incubation with 5 mM L-leucine methyl ester HC1 (Sigma Chemical Co.) in serum-free RPMI 1640 as described [23, 24]. The treated cell population was washed twice with medium RPMI and then incubated with neuraminidase-treated sheep red blood cells (SRBCs) [25]. The rosetting and nonrosetting populations were then separated by centrifugation on diatrizoateFicoll gradients. The nonrosetting cells obtained from the interface were incubated with 0.1 mM L-leucyl-leucine methyl ester HC1 in serum-free RPMI 1640 to delete residual NK

98

Journal

of Leukocyte

Biology

Volume

52, July

1992

with on The 2%

cells from the first centrifugation were treated with isotonic NH4C1 to lyse the SRBCs and then were passed over a nylon wool column to remove residual B cells and monocytes. Afterward, purified CD4 T cells were prepared by negative selection using a panning technique [26] to deplete contaminating HLA-DR-positive cells and CD8 cells. Cells were reacted with saturating concentrations of mAbs L243 plus OKT8 and, after washing, cells were added to goat antimouse Ig-coated panning dishes and incubated for 70 mm at 4#{176}C.Afterward, the nonadherent cells were gently aspirated and panned a second time on another GaMIg-coated petri dish. The nonadherent cells were harvested and found to contain < 0.1% esterase-positive cells, < 1% CD8-positive cells, and > 96% CD4-positive cells.

of Cell Culture

for T or B Cell Activation

Anti-CD3 mAb, 64.1, was diluted in Tris buffer (50 mM, pH 9.5) at a concentration of 4 zg/ml, and 250 tl were placed in each of the wells of 24-well macrotiter plates (Costar, Cambridge, MA) and incubated overnight at room temperature. The wells were washed with phosphate-buffered saline (PBS) to remove nonadherent mAbs and then 2-8 x 106 CD4 T cells were added to each well. Afterward, cells were incubated at 37#{176}Cin a humidified atmosphere of 5% CO2 and 95% air for varying periods of time. In some experiments, 5 x 106 highly purified B cells were added to cultures of CD4 T cells in wells coated with 64.1. In some experiments, B cells were cultured with SA and recombinant IL-2 but without T cells. These cells were stained with appropriate mAb followed by FACS or electron microscopic analysis.

Flow Cytometry Expression of CD11a, CD18, or CD54 was determined by staining cells with saturating concentrations of an appropriate mAb and counterstaining them with fluorescein isothiocyanate (FITh)-conjugated GaMIg (Cappel). The samples were then analyzed by flow cytometry using the FACScan system (Becton Dickinson).

Immunocytochemical

Purification

gradients of

cells were again rosetted SRBCs and centrifuged to remove residual T cells. B cells contained less than

esterase-positive monocytes and less than 1% T cells as determined by staining with OKT3 and OKT11 pan T cell mAb, followed by analysis with the fluorescence-activated cell sorter (FACS). The cells were also characterized as contaming more than 85% CD2O-positive B cells and no CD16-positive NK cells. The sedimented rosette-forming

Techniques

Hoffmann-La

treated

Study

by Electron

Culture plates were removed from the incubator to equilibrate at room temperature before the ing, all of which was performed on a rocker

Microscopy and allowed immunostaintable over ice.

Cells were stained with gold-conjugated RFB-4 (anti-CD22 mAb) for 60 minutes, washed three times in 2 % fetal calf serum (FCS) in RPM!, and fixed for 30 mm in 3% paraformaldehyde in 0.1 M phosphate buffer in order to abrogate passive antibody-induced redistribution and subsequent patching of cell surface molecules. After fixation, the cells were washed three times in 2% FCS, stained for 30 mm with biotinylated mAb R6.5 (anti-CD54 mAb) or a biotinylated control mAb (P1.17), washed three times in 2% FCS, and stained for 30 mm with horseradish peroxidase-streptavidin. Afterward, the cells were washed three times in PBS, fixed

r 30 mm nce in ubated

in half-strength

cacodylate for 15

minobenzidine. ashed once

Karnovsky’s

buffer mm in After the Tris buffer

in

fixative

and twice in Tris the dark in freshly 15-mm and

(27),

incubation, the twice in cacodylate

Length of Culture

washed

buffer, and prepared cells

T Cells

(hours)

indi-

were buffer,

B Cells

0

ed for 30 mm in 1% cacodylate buffer-osmium tetroxide, ashed in water, and dehydrated in an ethanol series. Cells rere removed from the plates with propylene oxide, plleted, nd embedded in Poly/Bed epoxy resin. Thin (900- A ) secions were then cut and viewed on a Philips 300 transmission lectron microscope. In some experiments, cells were not 6

xed with paraformaldehyde before the staining procedures n order to examine antibody-induced passive redistribution f cell surface molecules. To examine the expression and disribution of LFA-1 on the cell surfaces, a similar staining rocedure was carried out using mAb R7.1 (anti-CD11a TAb) or a control mAb (P1.17) and peroxidase-conjugated oat antimouse Ig followed by development with diminobenzidine. In all experiments the identity of T and B cells was ocumented by the presence of 15-nm gold particles conugated to the B cell-specific mAb RFB-4 (anti-CD22).

12

ESULTS

24

Expression of LFA-1 and ICAM-1 by Resting or Activated 3 Cells and CD4 T Cells: Analysis by FAGS ftc initial experiments examined the expression of LFA-1 tnd ICAM-1 on B cells and CD4 T cells by flow cytometry. B cells were activated with SA plus IL-2, and CD4 T cells were activated with immobilized anti-CD3 for varying periods of time. LFA-1 was expressed by approximately 75% Df resting B cells and 95% of resting CD4 T cells. The den-

48

C018

CD1Ia

72

Log Fluorescence Fig.

2.

CD4

Kinetics T cells.

ofthe B cells

expression and

ofCD54

CD4

T cells

lation or were activated for varying (anti-CD54) followed by Flit-conjugated of

a typical

Fig.

1. Kinetics

B cells

and

ately

after

R7.l

(anti-CD11a)

antimouse

ofthe

CD4

expression

T cells.

isolation

or activated

or 60.3

1g. The

results

findings,

64.1

(1 gig/well).

and

ofa

typical

and

CD4

for varying

(anti-CD18)

are shown. B cells titer plates with SA (1:60,000 (2 x l06/well) were activated mobilized

ofCDlla

B cells

Intensity

CD18

T cells periods

followed experiment,

by

resting

or activated

were analyzed immediof time and stained with by FITC-conjugated goat out

ofthree

with

the

same

(2 x l06/well) were activated in 24-well macrov/v) and IL-2 (50 U/ml), whereas CD4 T cells in 24-well macrotiter plates coated with im-

out

of

three

cells (2 (1:60,000 activated

x l06/well) were activated v/v) and IL-2 (50 U/mI), in 24-well macrotiter

gig/well).

Dotted

solid

Log Fluorescence

experiment,

lines

indicate

lines indicate CD54

or activated

B cells

analyzed

immediately

after

were

periods the

in 24-well whereas CD4 plates coated

control

and anti-mouse

of time

goat with

Intensity

by resting

staining

same

stained

with

1g. The

findings,

are

and iso-

R6.5 results

shown.

B

macrotiter plates with SA T cells (2 x 106/well) were with immobilized 64.1 (1

with P1.17 (control

mAb)

and

expression.

sity of LFA-1 on resting CD4 T cells was greater than that on resting B cells. The percentage and mean fluorescence intensity of the positively stained cells were increased by activation, such that both cell populations expressed LFA-1 comparably by 24 h (Fig. 1). ICAM-i molecules were expressed on approximately 55% of resting B cells but on fewer than 5% of resting CD4 T cells. Within 6 h of activation, however, there was a significant increase in the number of ICAM-1-positive T cells, whereas an increase in the number of ICAM-1-positive B cells required 12 h of activation. Thereafter ICAM-i expression ofboth CD4 cells and B cells continued to increase (Fig. 2).

Tohma

ci al.

T cell-B

cell

interactions

99

Expression and Distribution of LFA-1 and ICAM-1 on B Cells and CD4 T Cells: Analysis by Electron Microscopy The next experiments examined the expression and distribution of LFA-i or ICAM-1 on B cells and CD4 T cells in detail by electron microscopy. In these studies, B cells were identified by immunogold staining ofCD22, a B cell-specific molecule. Staining with mAb to CD22 has no effect on the density or distribution of B cell LFA-1 or ICAM-i (data not shown). The LFA-i molecules were uniformly distributed on the surface of CD4 T cells and B cells. The distribution of LFA-i remained uniform on each cell population after activation or after coculture of anti-CD3-stimulated CD4 T cells and B cells (Fig. 3). For comparison, there is no immunoperoxidase staining of anti-CD3-activated CD4 T cells stained with a control mAb of irrelevant specificity (Fig. 4). Similar negative staining was noted when T cell-B cell conjugates were stained with a control mAb. As can be seen in Figure 5, resting B cells expressed ICAM-1 molecules homogeneously on the cell surface,

I)

.-

.

(

L

B Fig.

4.

Lack

of immunoperoxidase

activity

associated

with

anti-CD3-acti-

vated lized

T cells stained with a control mAb. CD4 T cells activated by immobi64.1 for 24 h were stained with P1.17 and peroxidase-conjugated goat antimouse Ig followed by development with diaminobenzidine.

whereas resting not shown). The was not altered

S

T

Fig.

3.

cell-B (5

Uniform cell

x 106/well)

were

CD3

(64.1,

R7.l

(anti-CDlla)

100

of LFA-l

for

and with

conjugated

Journal

24

h.

CD4

T cells

cells (8 macrotiter

Afterward,

cells

peroxidase-conjugated

diaminobenzidine. to the

on

CD4 T in 24-well

activated

1 gig/well)

development des

expression collaboration.

B cell-specific

of Leukocyte

goat Note mAb,

Biology

the

and

during

T

cells anti-

were

with

immunostained

antimouse

presence

RFB-4

B cells

x l06/well) and B plates coated with Ig followed

of 15-nm

gold

by path-

(arrows).

Volume

52, July

1992

CD4 T cells did not express ICAM-1 (data distribution of ICAM-i expressed by B cells by activation or coculture with anti-

CD3-stimulated CD4 T cells (Fig. 6). By contrast, ICAM-1 molecules were highly mobile on the surface of activated CD4 T cells. Thus, if the activated CD4 T cells were not fixed, staining, even at 4#{176}C, induced redistribution of ICAM-1 into aggregates (Fig. 7). This phenomenon is consistent with the observation of other investigators of the passive, antibody-induced migration and subsequent patching of cell surface antigens on unfixed cells [28, 29] but has not previously been noted with ICAM-i. Moreover, differences in the capacity of mAb staining to induce redistribution of ICAM-1 on T cells and B cells has not previously been noted. When CD4 T cells were fixed before all staining procedures, the distribution of ICAM-1 molecules on CD4 T cells was uniform with no aggregation (Fig. 8). The final experiments were carried out to determine whether there was redistribution of ICAM-i molecules on activated CD4 T cells during coculture with B cells. To assess this accurately, cells were fixed with paraformaldehyde before staining procedures and then CD4 T cells that had been activated with immobilized anti-CD3, or CD4 T cells activated with immobilized anti-CD3 and cultured with B cells, were analyzed. When CD4 T cells were activated with immobilized anti-CD3 mAb in the absence of B cells, the

.

vated T cells plays an important role in T cell-B ration leading to B cell activation. In the current role of LFA-i-ICAM-1 interactions was studied

.

.

/

.,.

r.#{231}j

detail by examining adhesion molecules

the expression during T cell-B

goal was adhesion

the expression at the point

From

to visualize molecules results

of FACS

and distribution cell collaboration.

and distribution of T cell-B cell

analysis,

cell collabostudies, the in greater

resting

CD4

of these The

of these interaction. T cells

and

#{149}._J_

I

1

I

‘

-%-

I

B cells express LFA-1 molecules, indicating that both cell types could be ready to bind to counterreceptors after receiving activation stimuli that increase the avidity of LFA-1, because this activation step appears to be necessary for LFA-1 to bind to its ligands [34]. Furthermore, the density of LFA-1 on both cell populations was increased by activation, suggesting that lated by both

LFA-1-dependent the quantity

interactions could be avidity of LFA-1 expressed

and

reguby

the activated cells. Alterations in the density of LFA-1, however, do not appear to be important in T cell-B cell collaborations, because the density of LFA-1 did not increase substantially during the first 24 h of activation when the critical events of T cell-B cell collaboration occur [9]. On the other hand, resting B cells express ICAM-1 without activation as evidenced by both FACS and electron microscopic

analysis.

By

contrast,

CD4

T

cells

did

not

cx-

-‘

i:\:

‘V

.

.

#{149}#{149}

H. #{149}6

Fig.

5. Resting B cells express CD54 molecules uniformly on the cell surface. Resting B cells were immunostained with a colloidal gold-conjugated RFB-4 (anti-CD22) and biotinylated R6.5 (anti-CD54), followed by streptavidin-peroxidase. CD54 is uniformly distributed along the entire plasma membrane of B cells. Note 15-nm colloidal gold particles conjugated to the B cell-specific mAb, RFB-4, some of which are being internalized (arrow).

majority (95%; 17 of 20) exhibited an even distribution of ICAM-1 molecules (Fig. 8). However, when activated CD4 T cells were cocultured with B cells, the majority of CD4 T cells (85%; 17 of 20), but not B cells, redistributed ICAM-i such that it was found at or immediately adjacent to the point of attachment to the B cell (Fig. 6).

DISCUSSION In models of T cell-dependent shown that LFA-1/ICAM-1-dependent important

have and

role

previously ICAM-1

polyclonal

in

T

cell-B

B cell cell

reported that play an important

activation

of

human

collaboration

interactions role in B cells

j.s

activation, interactions

it has been play an [5-7, 9]. We between LFA-i T cell-dependent

induced

by

7

anti-CD3

stimulation of the T cells. Moreover, the orientation of this receptor-counterreceptor pair appeared to involve an interaction of ICAM-1 expressed by activated T cells with B cell LFA-1, because LFA-1-negative T cell clones from a patient with leukocyte adhesion deficiency retained the ability to induce B cell activation in an ICAM-1-dependent manner [30]. A role for engagement of LFA-1 in B ce1 activation is supported by the observation that these structures can function as signaling molecules [31-33]. Thus, it is likely that the engagement of B cell LFA-i by ICAM-1 expressed on acti-

Fig. 6. CD54 on activated CD4 T cells, but not B cells, is localized at and about the point of attachment to B cells. CD4 T cells (8 x l06/well) and B cells (5 x 106/well) were activated in 24-well macrotiter plates coated with anti-CD3 (64.1, 1 gig/well) for 24 h. Afterward, cells were immunostained with a colloidal gold-conjugated RFB-4 (anti-CD22) and biotinylated R6.5 (anti-CD54), followed by streptavidin-peroxidase. Paraformaldehyde fixation preceded staining with biotinylated R6.5. CD54 is uniformly distributed on the B cell surface, but CD54 on activated CD4 T cells is localized around the point ofattachment ofthe B cells. Note 15-nm colloidal gold particles conjugated to the B cell-specific mAb, RFB-4 (arrow).

Tohma

et al.

T cell-B

cell

interactions

101

);A#{176}

fore, newly synthesized ICAM-i may exhibit different properties than constitutively expressed molecules. Clearly, however, the basis for differences in the biologic behavior of T cell ICAM-1 and B cell ICAM-i remains to be completely delineated. It has been shown by several investigators that LFA-1 molecules might function not only to promote adhesive interactions but also as signaling molecules [31-33]. It, therefore, is possible that during T cell-B cell collaboration engage-

4.

.

...J1 ,.

\

‘.

..

.,

ment than

41 .

iF

t...

.

...-

.

“\

I

,-

-

,

.

-;.

Fig. 7. Immunostaining on activated, unfixed by immobilized 64.1 (anti-CD54), followed

of CD54 causes redistribution CD4 T cells into aggregates. for 24 h were immunostained by streptavidin-peroxidase.

CD4 with

of CD54 molecules T cells activated biotinylated R6.5

of LFA-1 transmits merely promoting

activation heterotypic

signals adhesion.

to B cells rather Alternatively,

LFA-1-ICAM-1 interactions may promote cell-to-cell contact, permitting signal transduction through other surface molecules. In either case, the redistribution of ICAM-1 molecules on activated CD4 T cells to the site of contact with B cells could play a central role in T cell-B cell collaboration. Uniform distribution of LFA-i on both B cells and CD4 T cells was found under the experimental conditions examined, including resting and activated states or coculture with immobilized anti-CD3. It was somewhat surprising that capping of LFA-1 on B cells the aggregation of ICAM-i possible that the capacity

LFA-1 and to cap or

was on

not observed in parallel activated CD4 T cells.

ICAM-i have intrinsic that B cells cap these

with It is

differences molecules

in less

press ICAM-1. Activation increased the expression of ICAM-i on both CD4 T cells and B cells. An increased density of ICAM-i by CD4 T cells could be seen within 6 h of activation, whereas B cells increased expression of ICAM-1 by 12 h. These findings suggest that the interaction between CD4 T cells and B cells might be dependent on the density of ICAM-1 expressed by activated CD4 T cells in this system. Alternatively, because the affinity of T cell LFA-1 for ICAM-1 can be transiently up-regulated by stimulation with anti-CD3 [34], it is possible that these cells could then bind ICAM-1 constitutively expressed by resting B cells. However, we have previously noted that LFA-i-negative T cell clones from a patient with leukocyte adhesion deficiency have the ability to activate B cells in this system, so it is unlikely that an adhesive interaction between T cell LFA-1 and B cell ICAM-i is necessary for polyclonal B cell activation [9]. Although not necessary, LFA-1 expressed by activated CD4 T cells may play a role in the initiation of T cell-B cell collaboration, as we have found that mAb to LFA-i expressed by activated fixed T cells partially blocks the induction of B cell activation. The most striking finding in the current study is the redistribution of ICAM-1 on activated CD4 T cells but not B cells. There are several possible ways to explain the localization of T cell ICAM-1 to regions of contact with B cells. It is possible that activated T cells might recognize the site of contact with B cells and express ICAM-1 molecules at site. Alternatively, and more likely, ICAM-1 molecules

I

.

.

(

this cx-

pressed by activated CD4 T cells might bind to LFA-i molecules on B cells and then redistribute the site of contact with B cells. In either case, it seems that the redistribution of ICAM-i on activated CD4 T cells to the site of contact with B cells might play an important role in T cell-dependent B cell activation, because fixed activated T cells, which cannot redistribute ICAM-1, do not induce B cell responses as well as live cells, even in the presence of exogenous T cell-derived lymphokines [30]. The explanation for differences in the capacity to aggregate between B cell ICAM-i and T cell ICAM-1 remains unclear. It is possible that differences in the behavior of the molecules are related to the observation whereas

102

that ICAM-i it must by

Journal

is constitutively synthesized de

of Leukocyte

Biology

expressed novo by T

Volume

on cells.

52,

July

B cells, There-

1992

Fig. CD4

8. CD54

is uniformly

T cells activated

distributed

on

by immobilized

64.1

with biotinylated R6.5 (anti-CD54), Activated CD4 T cells were fixed ing with biotinylated R6.5.

activated, for

fixed

CD4

T cells.

24 h were immunostained followed by streptavidin-peroxidase. with 3% paraformaldehyde before stain-

ifectively than activated )ing of LFA-1 occurred

T cells. It is also possible that caplater in culture and thus was not ob-

erved. This possibility appears unlikely because the relevant nteractions between LFA-1 and ICAM-1 occur before the ime of the ultrastructural analysis. Finally, a difference in he relative amounts of LFA-1 and ICAM-i expressed on :ells might explain this finding. Thus, LFA-1 molecules are :xpressed at much greater density than ICAM-i molecules, ;o it may be difficult to detect redistribution or aggregation )f a small percentage of LFA-1 molecules by the cells. In summary, the increased expression of ICAM-1 and the edistribution of ICAM-1 by activated T cells during human I cell-dependent B cell collaboration suggest that T cell CAM-i may deliver an important signal leading to B cell tctivation.

ICKNOWLEDGMENT rhis rants

work

was AR-09989

supported by and AR-39169.

U.S.

Public

Health

Service

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