Expression and distribution of CD11aICD18 and CD54 during human T cell-B cell interactions

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: 97-103; intercellular adhesion function-associated . T cell-B cell 1992. antigen molecule 1(LFA-1) 1 In the 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 surface current studies show that of activated CD4 T cells ICAM-1 is dynamic expression on the and highly mo- bile. Thus, B cells exhibit a uniform distribution under the experimental conditions examined. when activated CD4 T cells were cocultured redistribution of T cell ICAM-1 to the point to the B cell was observed. These results suggest of ICAM-1 T cell-B of CD4 T cells cell collaboration. may of ICAM-1 By contrast, with B cells, of attachment that redistriplay a role in CDJJaJCDJ8 Abbreviations: fluorescence-activated immune role Medicine, interaction INTRODUCTION portant during E. Lipsky of Internal bution facilitating (ICAM-1) 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 all leukocytes. Interactions j32 integrin mediated family, is expressed by 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) METHODS cells [24]. The neuraminidase-treated diatrizoate-Ficoll resulting population Antibodies mAbs were used, including OKTi1 (American Collection [ATCC]), an immunoglobulin mAb directed at the CD2 molecule on Type G2a cells; T OKT3 (A’ICC) and 64.1, IgG2a mAbs directed at the CD3 molecular complex on mature T cells [15, 16]; OKT8 (ATCC), an IgG2a mAb directed at the CD8 molecule; R7.1 (a gift of Dr. Robert Rothlein, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT), an IgGi mAb directed at CD11a; 60.3 (a gift of Dr. Patrick Beatty, Fred Hutchinson Cancer Center, Seattle, WA), an IgG2a mAb directed at CD18; RFB-4 (a gift ofDr. Ellen Vitetta, University of Texas Southwestern Medical Center at Dallas, Dallas, TX), an IgGi mAb directed at CD22 on B cells; 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 (PBMCs) were obtained from healthy adult volunteers by centrifugation heparinized venous blood over sodium diatrizoate-Ficoll gradients (Sigma Chemical Co., St. Louis, MO) [21]. Cell of Purification 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 gradients of cells were again rosetted SRBCs and centrifuged to remove residual T cells. B cells contained less than with on The 2% 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 cells from the first centrifugation NH4C1 to lyse the SRBCs and wool column to remove residual were treated with isotonic then were passed over a nylon B cells and monocytes. Af- terward, 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. Techniques Hoffmann-La treated 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 Study by Electron Microscopy Culture plates were removed from the incubator and allowed to equilibrate at room temperature before the immunostaining, all of which was performed on a rocker table 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 in half-strength Karnovsky’s fixative (27), Length of Culture washed xed with paraformaldehyde before n order to examine antibody-induced f cell surface molecules. To examine to the B cell-specific 6 the staining procedures passive redistribution the expression and dis- mAb RFB-4 B Cells 0 ribution 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 T Cells (hours) nce in cacodylate buffer and twice in Tris buffer, and inubated for 15 mm in the dark in freshly prepared diminobenzidine. After the 15-mm incubation, the cells were ashed once in Tris buffer and twice in cacodylate buffer, 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 12 (anti-CD22). 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 ofthe CD4 ately after R7.l (anti-CD11a) isolation antimouse findings, titer plates (2 mobilized or activated shown. with x l06/well) 64.1 ofCDlla B cells or 60.3 1g. The are expression T cells. results (1 gig/well). CD4 for varying (anti-CD18) ofa B cells SA (1:60,000 were activated and typical (2 Intensity and periods followed experiment, x l06/well) CD18 T cells were by resting or activated were analyzed immediof time and stained with by FITC-conjugated goat out ofthree activated v/v) and IL-2 (50 U/ml), in 24-well macrotiter with in 24-well whereas plates CD4 coated the same macroT cells 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. vated lized 4. Lack of immunoperoxidase antimouse Ig followed whereas resting not shown). The was not altered 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 RFB-4 B cells during T x l06/well) and B plates coated with cells anti- were with immunostained antimouse presence Ig followed of 15-nm gold by path- (arrows). Volume associated with anti-CD3-acti- by development with diaminobenzidine. 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 S T activity 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 52, July 1992 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 activated with immobilized anti-CD3 and cultured cells, were analyzed. When CD4 T cells were activated immobilized anti-CD3 mAb in the absence T cells with B with of B cells, the . vated T cells plays an important role in T cell-B cell collaboration leading to B cell activation. In the current studies, the role of LFA-i-ICAM-1 interactions was studied in greater detail by examining the expression and distribution of these adhesion molecules during T cell-B cell collaboration. The . . / .,. goal was adhesion r.#{231}j From to visualize molecules results the expression at the point of FACS and distribution of T cell-B cell analysis, resting CD4 of these interaction. T cells and #{149}._J_ I 1 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 gesting that lated by both the activated however, do laborations, I populations LFA-1-dependent the quantity was increased and interactions could be avidity of LFA-1 expressed by activation, sugreguby cells. Alterations in the density of LFA-1, not appear to be important in T cell-B cell colbecause 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 ized around the point ofattachment ofthe particles conjugated to the B cell-specific Tohma et al. on activated CD4 T cells is localB cells. Note 15-nm colloidal gold mAb, RFB-4 (arrow). T cell-B cell interactions 101 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 );A#{176} delineated. It has been molecules might actions but also is possible that 4. . ...J1 ,. \ ‘. .. ., ment than 41 . iF ...- . I “\ ,- - , . -;. Fig. 7. Immunostaining on activated, unfixed CD4 by immobilized (anti-CD54), for 24 h were immunostained by streptavidin-peroxidase. 64.1 followed of CD54 causes redistribution of CD54 T cells into aggregates. CD4 T cells with molecules activated biotinylated 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 t... . shown by several investigators that LFA-1 function not only to promote adhesive interas signaling molecules [31-33]. It, therefore, during T cell-B cell collaboration engage- R6.5 capping of LFA-1 the aggregation possible that the capacity on B cells was not observed in parallel of ICAM-i on activated CD4 LFA-1 and ICAM-i have intrinsic to cap or that B cells cap these with T cells. differences molecules It is 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]. I 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 is possible that activated to regions T cells of contact with might recognize B cells. the site . . ( It of contact with B cells and express ICAM-1 molecules at this site. Alternatively, and more likely, ICAM-1 molecules cxpressed 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 by immobilized with biotinylated R6.5 (anti-CD54), Activated CD4 T cells were fixed with ing with biotinylated R6.5. on activated, for fixed CD4 T cells. 24 h were immunostained followed by streptavidin-peroxidase. 3% paraformaldehyde before stain64.1 ifectively than activated T cells. It is also possible that cap)ing of LFA-1 occurred later in culture and thus was not oberved. 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 EFERENCES 1. Davignon, D., Martz, E., Reynolds, T., Kurzinger, K., and Springer, TA. Lymphocyte function-associated antigen one (LFA-l): a surface antigen distinct from Lyt2, 3 that participates in T lymphocyte-mediated killing. Proc. Nail. Acad. Sci. USA. 78, 4535, 1981. 2. Davignon, D., Martz, E., Reynolds, T, Kurzinger, K., and Springer, TA. Monodonal antibody to a novel lymphocyte function-associated antigen (LFA-l): mechanism of blockade of T lymphocyte-mediated killing and effects on other T and B lymphocyte functions. j Immunol. 127, 590, 1981. 3. 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