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Human Effector Memory T Cells Express CD86: A Functional Role in Naive T Cell Priming

Centre d’Immunologie Pierre Fabre, Saint-Julien en Genevois, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The glycoprotein CD86 expressed on APCs provides a costimulatory signal necessary for an efficient activation of naive T cells. In contrast, there is controversy about the condition of expression and the function of CD86 on T cells. In this study, we have analyzed the phenotype and the biological activity of CD86⁺ T cells generated from human PBMC. Results show that CD86 expression on T cells is induced by long term stimulation via CD3 and IL-2R and is down-regulated as the cells become quiescent. The CD86-expressing cells are memory effector T cells: 1) they express CD45RO and high levels of the activation markers CD25, CD54, and HLA-Dr; 2) they selectively express CD30, CD40-ligand, and CD70; and 3) in response to stimulation, most of them produce IFN- before dying by apoptosis. We then analyzed whether CD86 expressed on T cells is functional. Results show that paraformaldehyde-fixed CD86⁺ T cells enhance the proliferation and production of IFN- by anti-CD3 mAb-stimulated naive T cells and induce proliferation of resting allogenic T cells. All these effects are prevented by neutralizing anti-CD86 mAbs. In contrast, we report no autocrine effect of CD86 in CD86⁺ T cell activation. In conclusion, these data show that human memory effector T cells express a functional form of CD86 that can costimulate naive T cell responses.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Naive T cells require primary and costimulatory signals from APCs to be functionally activated. The primary signal, provided by the CD3/TCR complex, initiates activation. The costimulatory signal, provided by cell surface interactions, induces clonal expansion and differentiation into effector cells 1, 2 . Among the different molecules with costimulatory properties, CD28 appears the most potent. The interaction of CD28, expressed on resting and activated T cells, with the B7 molecules, CD80 (B7.1) and CD86 (B7.2), results in an increase in T cell proliferation, cytokine production, and resistance to apoptosis 3, 4, 5 . In contrast, T cells that bind the Ag and that do not receive a costimulatory signal are thought to die or to become anergic 1, 2, 5 . In addition to CD28, some populations of T cells may also transiently express CTLA-4, a second ligand for CD80 and CD86. CTLA-4 engagement may transduce an "off" signal, disengaging T cells from further activation and proliferation 5, 6 . As such, T cell activation is determined by a delicately balanced interplay between positive signals from CD28 and negative signals from CTLA-4.

Numerous APC express the B7 molecules either constitutively or in response to stimulation 3 . The expression of these molecules has been also reported on T cells 3, 7, 8, 9, 10, 11, 12, 13, 14 . Activated, but not resting, human T cells express functional CD80 that is involved in T-T cell interaction 7, 8, 9 . In contrast, the condition of the expression and the function of CD86 on T cells is debated. Although some freshly isolated murine T cells express CD86 10, 14 , resting human T cells do not 3 . Activation down-regulates CD86 expression on murine T cell clones (TCC)², 11 , whereas it induces CD86 expression on human T cells 3 . Furthermore, CD86 transfected in a murine T cell line 12 or expressed on human TCC fails to provide costimulatory signal through CD28 13 . Moreover, CD86 on human TCC appears in an hypoglycosylated form 13 . Nevertheless, it has been recently shown that CD86 on fresh murine T cells was functional 14 . As such, we have investigated herein the generation and the function of normal, nontransformed CD86⁺ human T cells.

	Materials and Methods

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Generation of CD86-expressing T cells

PBMC were isolated from blood from healthy volunteers by centrifugation on Ficoll/Paque (Pharmacia, Upsalla, Sweden). PBMC (10 x 10⁶/ml), cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (all from Life technologies, Cergy Pontoise, France) were stimulated with 200 ng/ml anti-CD3 mAb (clone OKT3; American Type Culture Collection, Manassas, VA) plus 20 ng/ml IL-2 (R&D Systems, Abingdon, U.K.). IL-2 (20 ng/ml) was added every 4 days. In some experiments, T cells were restimulated with anti-CD3 mAb plus IL-2 after 3 wk.

Cell surface labeling

The FACS analyses were performed using a FACSvantage cytofluorometer (Becton Dickinson, Erembodegem, Belgium) with the following mAbs: FITC-labeled anti-CD2 mAb, FITC- and phycoerythrin (PE)-labeled anti-CD3 mAbs, FITC-labeled anti-CD45RO, -CD54, -CD80, -HLA-Dr (all from Becton Dickinson), -CD70 (PharMingen, San Diego, CA), -CD30 (Dako, Glostrup, Denmark), or -CD40-ligand (CD40-L) (Ancell, Bayport, MN) mAbs and biotin-labeled anti-CD86 mAb (clone IT2.2) revealed by Cy-chrome-labeled streptavidin (both from PharMingen). Control isotype mAbs were from Becton Dickinson.

Simultaneous measurement of membrane CD86 and intracellular IFN- expression

Three weeks after the initial stimulation, T cells were restimulated for 6 h with 10 ng/ml PMA (Sigma, St. Louis, MO) plus 1 µM ionomycin (Calbiochem, San Diego, CA) in the presence of 2.5 µg/ml brefeldin A (Molecular Probes, Eugene, OR). Cells were then stained with biotin-labeled anti-CD86 mAb revealed by Cy-chrome-labeled streptavidin. After washing, cells were fixed and permeabilized using the Cytoperm/cytowash kit (PharMingen) according to the manufacturer’s recommendations before staining with FITC-labeled anti-human IFN- (PharMingen) or control mouse IgG1 (Becton Dickinson) mAbs. Results are expressed in percent of IFN--expressing cells among the CD86^- and CD86⁺ T cell populations.

Simultaneous measurement of membrane CD86 expression and apoptosis

Three weeks after stimulation, T cells were restimulated for 24 h with 200 ng/ml anti-CD3 mAb. Then, T cells were stained with biotin-labeled anti-CD86 mAb revealed by allophycocyanin-labeled streptavidin (Molecular Probes), with propidium iodide and with FITC-labeled annexin V, using annexin V kit (Immunotech, Marseille, France) according to the manufacturer’s recommendations. Cells were analyzed using a FACSvantage cytofluorometer equipped with two lasers. Annexin V-FITC and propidium iodide emissions (530 nm and 630 nm, respectively) were measured after excitation by an Argon ion laser tuned at 488 nm (Coherent, Santa Clara, CA). Allophycocyanin emission (660 nm) was measured after excitation of an Helium Neon laser (Spectra Physics, Mountain View, CA) tuned at 633 nm. Results are expressed in percent of annexin-V-positive cells in the CD86^- and CD86⁺ T cell populations.

Western blotting analysis

Western blot analysis was performed with the following human cells: CD86⁺ T cells collected 1 wk after restimulation with anti-CD3 mAb plus IL-2, the Jurkat T cell line, the B cell lines Daudi and RPMI 8226 (all the cell lines are from American Type Culture Collection), and tonsillar B cells stimulated for 2 days with IL-4 plus anti-CD40 mAb, as described 15 . Cells were washed in ice-cold PBS before lysis in 10 mM phosphate buffer (pH 7.4) containing 0.5% Nonidet P40 (Sigma) and protease inhibitors (Boehringer Mannheim, Mannheim, Germany). Proteins from 5 x 10⁶ cells were electrophoretically separated on a 10% polyacrylamide gel in reducing conditions and then transferred on a nitrocellulose membrane (Bio-Rad, Ivry sur Seine, France). After saturation, membranes were incubated with a goat IgG anti-human CD86 Ab (R&D Systems). After washing, membranes were incubated with peroxydase-labeled affinity-purified rabbit anti-goat IgG Ab (Dako) and bound Abs were detected using the enhanced chemiluminescence system (Amersham, Amersham, U.K.).

In vitro costimulatory assay

The CD86⁺ T cells were collected 2 wk after restimulation with anti-CD3 mAb plus IL-2. As control, CD86^- T cells were isolated 2 wk after the initial stimulation by removing the CD86⁺ T cells by FACS sorting. These two populations contained only T cells; all the cells were labeled with a FITC-labeled anti-CD2 mAb, but not with FITC-labeled anti-CD14 and -CD20 mAbs. The CD86⁺ and CD86^- T cells were fixed with 1% paraformaldehyde (PFA) for 10 min, quenched with 0.2 M L-lysine, and washed three times before use. Naive (CD45RA⁺) CD4⁺ T cells have been purified from PBMC by sheep RBC rosetting followed by a negative selection using anti-CD8 and -CD45RO mAbs (both from Sigma) and anti-mouse Ig Ab-coated magnetic beads (Dynal, Oslo, Norway); the purity, determined by double color FACS analysis with FITC-labeled anti-CD45RA and PE-labeled anti-CD4 mAbs (both from Becton Dickinson), was >90%. CD45RA⁺ T cells have been stimulated with suboptimal concentration (10 ng/ml) of anti-CD3 mAb in the presence or absence of PFA-fixed CD86⁺ or CD86^- T cells in round-bottom 96-well culture plates (Nunc, Roskilde, Denmark), in quintuplicate. In proliferation assays, CD86^- or CD86⁺ fixed T cells and naive T cells have been used at 2.5 x 10⁵ cells/well. After 3 days, cells were pulsed with 0.25 µCi/well [³H]thymidine (Amersham) for 6 h. Radioactive incorporation was measured by standard liquid scintillation counting. Results are given in cpm or in stimulation index (SI) calculated as follows: A/O, in which A and O are the cpm values obtained when cells are cultured or not with fixed T cells, respectively. For the quantification of IFN- production, CD86^- or CD86⁺ fixed T cells and naive T cells have been used at 10⁶ cells/well. IFN- was quantified in the 48 h supernatants using specific mAbs from PharMingen, according to the manufacturer recommendations. Results are expressed in ng/ml or in percent of increase calculated as follows: (A - O/O) x 100, in which A and O are the IFN- levels obtained in the presence or absence of fixed T cells, respectively. In some experiments, 10 µg/ml of the neutralizing anti-CD86 mAbs IT2.2 (IgG2b) (PharMingen), FUN-1 (IgG1; Ancell), or the isotype control mAbs (PharMingen) were added. Results are expressed in percent of decrease: (A - O/A) x 100, in which A and O are the values obtained in the absence or presence of a neutralizing anti-CD86 mAb, respectively.

Primary allogenic MLR

Naive CD4⁺ T cells have been isolated from PBMC of four donors as described above and have been cultured at 2.5 x 10⁵ cells/well with 2.5 x 10⁵ cells/well PFA-fixed CD86⁺ or CD86^- T cells, in quintuplicate, in round-bottom 96-well culture plates. In some experiments performed with PFA-fixed CD86⁺ T cells, 10 µg/ml of the neutralizing anti-CD86 mAbs IT2.2 and FUN-1 or of the isotype control mAbs have been added. After 6 days, cells were pulsed with 0.25 µCi/well [³H]thymidine for 6 h. Radioactive incorporation was measured by standard liquid scintillation counting, and results are given in cpm values or in percent of decrease, calculated as described above.

Statistical analysis

Statistical analyses were performed using the Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation and phenotyping of human CD86⁺ T cells

The expression of CD86 on human T cells is induced by stimulation. The expression of CD86 (Fig. 1a) and of the corresponding mRNA (data not shown) is undetectable on freshly isolated peripheral blood T cells 3, 5 . Stimulation via CD3 and IL-2R induces CD86 expression on some T cells; the percentage of CD86⁺ T cells is 20 ± 3% (mean ± SD, n = 7) 1 wk after stimulation and reaches a maximum after 3 wk (61 ± 25%) (Fig. 1a). When restimulated at day 21, all of the T cells become CD86⁺ within 1 wk for 1–2 wk (Fig. 1a). In the absence of restimulation, CD86 expression is down-regulated as the cells become quiescent (Fig. 1a).

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Stimulated human T cells express CD86. a, Kinetics of induction of CD86 expression on T cells. PBMC have been stimulated with anti-CD3 mAb plus IL-2 and have () or have not () been restimulated 3 wk later. The expression of CD86 on T cells has been analyzed weekly by FACS using FITC-labeled anti-CD3 mAb and biotin-labeled anti-CD86 mAb revealed by Cy-chrome-labeled streptavidin. Results are expressed in percent of CD86+ cells among the CD3+ T cells and are representative of one of seven experiments. b, CD86 expression is restricted to memory T cells. Two weeks after the initial stimulation, PBMC were stained with PE-labeled anti-CD3, FITC-labeled anti-CD45RO, and biotin-labeled anti-CD86 mAbs revealed by Cy-chrome-labeled streptavidin. Cells were gated on CD3+ T cells. One representative experiment of five is shown. c, CD30 expression is restricted to CD86+ T cells. Three weeks after stimulation, the expression of CD30 on CD86+ vs CD86- memory T cells has been evaluated by FACS using FITC-labeled anti-CD30 mAb and biotin-labeled anti-CD86 mAbs revealed by Cy-chrome-labeled streptavidin. One representative experiment of five is shown.

We have analyzed by FACS the phenotype of the CD86⁺ T cells. Results show that CD86 expression is restricted to CD45RO⁺ (memory) T cells (Fig. 1b). After stimulation, the percentage of CD45RO⁺ cells increases in a time-dependent manner, with all of the cells being CD45RO⁺ after 3 wk (data not shown). At this time point, we have compared the phenotype of CD86⁺ T cells with those of CD86^- T cells. Results show that CD86⁺ T cells express higher levels of the activation markers CD25, CD54, and HLA-Dr and that some of them selectively express CD30 and CD70 (Fig. 1c and Table I). A transient expression of CD40-L is also observed on some CD86⁺ T cells (Table I). As expected, stimulation also induces CD80 on T cells 7, 9 . The expression of CD80 is not restricted to a subset of T cells (Table I) and is more transient than CD86 expression, which is detectable 3 wk after stimulation; CD80 expression disappears within 1 wk in the absence of restimulation (data not shown) 7, 8, 9 .

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Biological activity of CD86+ T cells. a, Intracellular measurement of IFN- in CD86- and CD86+ T cells. Three weeks after the initial stimulation, T cells were restimulated for 6 h with PMA plus ionomycin in the presence of brefeldin. After staining with biotin-labeled anti-CD86 mAb, revealed by Cy-chrome-labeled streptavidin, cells were fixed, permeabilized, and labeled with specific mAb to detect intracellular IFN- (a) or with isotype control mAb (b). One representative experiment of five is shown. b, Activation-induced cell death measurement in CD86- and CD86+ T cells. Three weeks after the first stimulation, T cells were restimulated with anti-CD3 mAb for 24 h. After staining with biotin-labeled anti-CD86 mAb, revealed by allophycocyanin-labeled streptavidin, cells were labeled with FITC-annexin V and with propidium iodide. Cells were gated on CD86- (c) and CD86+ (d) T cells. One representative experiment of five is shown.

The glycoprotein CD86 expressed on human B and T cells is identical. Based on the observation that CD86 expressed on TCC had an apparent molecular mass of 70 kDa, whereas CD86 on CD86-transfected Chinese hamster ovary cells or on EBV-transformed B cells had a molecular mass of 90 kDa, it has been reported that CD86 expressed on human T cells was hypoglycosylated 13 . We show here that CD86 expressed on CD86⁺ T cells, IL-4 plus anti-CD40 mAb-stimulated tonsillar B cells, and the Daudi cells run with the same apparent molecular mass of 70 kDa (Fig. 3 and 3 . Furthermore, sequencing shows that CD86 expressed by T cells is homologous to the nucleic acid sequence reported in the GenBank database (data not shown). Nevertheless, we report that CD86 on RPMI 8866 B cells has a higher molecular mass of 90 kDa (Fig. 3). As a control, CD86 expression is undetectable on the Jurkat T cells (Fig. 3 and 13 . These results show that human T and B cells express the same isoform of the glycoprotein CD86.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. CD86 expressed on human T and B cells has a molecular mass of 70 kDa. Whole lysates from the Daudi human B cell line (lane 1), human tonsillar B cells stimulated for 2 days with IL-4 plus anti-CD40 mAb (lane 2), CD86+ T cells obtained 1 wk after restimulation (lane 3), the human B cell line RPMI 8866 (lane 4), and the human T cell line Jurkat (lane 5) have been examined for CD86 expression by Western blot analysis in reducing condition using polyclonal IgG anti-CD86 Ab.

We also evaluated whether CD86 expressed on T cells is functional. CD86⁺ T cells, obtained 2 wk after restimulation, have been fixed with PFA and used either as costimulators in anti-CD3 mAb assays or as stimulators in primary allogenic MLR. CD86^- T cells have been purified 2 wk after the initial stimulation, fixed with PFA, and used as control. CD80 expression was undetectable by FACS analysis on CD86⁺ and CD86^- T cells (data not shown).

In anti-CD3 mAb assays, naive CD4⁺ T cells have been stimulated with a suboptimal concentration of anti-CD3 mAb in the absence or presence of fixed CD86⁺ or CD86^- T cells. Results from five separate experiments show that the presence of CD86⁺ T cells potentiates anti-CD3 mAb-induced proliferation (SI = 7.2 ± 2, mean ± SD, n = 5) and IFN- production (increase of 580 ± 5%, mean ± SD, n = 5). Both of these effects are partly prevented by adding the neutralizing anti-CD86 mAbs, FUN-1 (decrease of 80 ± 7% and 78 ± 18%, respectively, mean ± SD, n = 5) or IT2.2 (decrease of 72 ± 5% and 67 ± 10%, respectively). In contrast, fixed CD86^- T cells are ineffective. The results from a representative experiment are shown in Fig. 4.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. CD86 expressed on T cells potentiates anti-CD3 mAb-induced naive T cell activation. Naive (CD45RA+) CD4+ T cells have been stimulated with 10 ng/ml anti-CD3 mAb in the absence or presence of PFA-fixed CD86+ or CD86- T cells. In some experiments, the neutralizing anti-CD86 mAbs, IT2.2, or FUN-1 or isotype control mAbs (IgG2b and IgG1, respectively) were added. a, Naive T cell proliferation was evaluated at day 3. b, IFN- production was quantified in the 48-h supernatants by ELISA. a and b, Results are expressed in mean ± SD of quintuplicate values and are representative of one of five separate experiments.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. CD86+ T cells stimulate a primary alloantigen MLR. Naive T cells from two different donors ( and ) have been cultured with PFA-fixed CD86+ T cells in the presence or absence of the neutralizing anti-CD86 mAbs, IT2.2, or FUN-1 or of the isotype control mAbs (IgG2b and IgG1, respectively). As controls, T cells have been cultured alone or with PFA-fixed CD86- T cells. T cell proliferation was measured at day 6. Results are expressed in cpm, mean ± SD of quintuplicate values and are representative of the data obtained with two of four donors.

Taken together, these data suggest that CD86 expressed on human memory T cells is functional.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We show here that the stimulation of human peripheral blood T lymphocytes induces the generation of effector memory cells expressing CD86. In vitro data suggest that CD86 expressed on T cells is functional and can costimulate naive T cell responses.

Our results show that the expression of the costimulatory molecule CD86 on T cells varies with their state of activation. CD86 expression is induced on memory T cells by stimulation and is down-regulated as the cells become quiescent. In contrast, it has been reported that activation down-regulates CD86 expression on murine TCC 11 . As such, the regulation of CD86 expression may differ in human and mouse T cells. Nevertheless, our data are in agreement with a previous study showing that CD86 is detectable on human peripheral blood T cells 10 days after stimulation 3 .

In agreement with these observations, we report that CD86⁺ T cells express high levels of activation markers and selectively express the cell surface molecules CD30, CD40-L, and CD70, whose expression is activation dependent 2, 18, 19 . More precisely, CD30 expression on T cells has been shown to be dependent on the presence of exogenous IL-4 or on CD28 triggering 20 . In agreement with others 21 , we report the generation of CD30⁺ T cells in the absence of exogenous IL-4. Nevertheless, because CD86 expression on T cells occurs earlier than CD30 expression (data not shown), CD30 expression could be induced through an autocrine CD86-CD28 interaction. Although controversial 21 , it has been also proposed that CD30 expression could be restricted to IL-4-producing T cells 22 . However, we find that CD30 expression is restricted to CD86⁺ T cells and that most of the CD86⁺ T cells produce IFN-. Moreover, in additional experiments, we have observed that human Th1, Th2, and Th0 TCC express CD86 (data not shown). As such, CD86 does not appear as a marker specific for T cells producing preferentially Th1 vs Th2 lymphokines.

Previous reports have shown that although freshly isolated T cells are resistant to apoptosis, the sensitivity to anti-CD3 mAb triggered cell death gradually increases upon activation and IL-2-dependent culture of T cells 16 . In agreement with these observations, we find that CD86⁺ T cells are highly sensitive to anti-CD3 mAb-triggered apoptosis. Until now, the mechanism(s) that controls the death/survival of long term stimulated memory T cells has not been completely understood 2 . Using a Fas-Fc molecule, we only partly prevented anti-CD3 mAb-induced CD86⁺ T cell death (data not shown). Moreover, addition of a neutralizing anti-CD86 mAb to CD86⁺ T cells does not modulate anti-CD3 mAb-induced apoptosis or lymphokine production, thereby showing that an autocrine T-T interaction between CD86 and CD28 or CTLA-4 that can be transiently expressed after activation does not modulate T cell activation.

Collectively, these findings show that CD86⁺ T cells express high levels of activation markers, produce high levels of cytokines, are sensitive to activation-induced cell death, and are poorly dependent of costimulatory signals to be efficiently activated, suggesting that CD86⁺ T cells are effector memory T cells.

The CD86 molecule expressed on human NK cells and Daudi B cells has been initially described as an 70 kDa glycoprotein 3 . Recently, it has been reported that CD86 on human TCC was hypoglycosylated based on the observation that CD86 on EBV-infected human B cell lines and on CD86-transfected Chinese hamster ovary cells had 90 kDa whereas CD86 on TCC was 70 kDa 13 . Our data also suggest that CD86 may present different levels of glycosylation according to the cell types in which it is expressed. Nevertheless, we report that both activated human B cells, which are professional APC, and effector memory T cells express a CD86 molecule of 70 kDa.

We report here that CD86 expressed on effector memory T cells has costimulatory properties. In contrast, it has been reported that CD86 on human TCC was not functional because it failed to provide a costimulatory signal to another TCC 13 . It has been now extensively reported that T cell response to stimulation is dependent on the strength of the T cell-APC interaction and on the status of T cell activation, resting naive T cells having more stringent requirements than effector memory cells 1, 2, 23 . TCC are effector memory cells that are poorly dependent of costimulatory signals to be efficiently activated. As such, it could be hypothesized that authors have reported no costimulatory effect of CD86 on human TCC due to the use of TCC as responder T cells 13 . The observation that CD86 on effector memory T cells does not influence their own activation reinforces this hypothesis. Reciprocally, using memory T cells as responders, others have suggested that the T-T cell interaction between CD2 and CD58 was involved in T cell activation 13, 24 . We found here that CD58 is expressed at similar levels on CD86⁺ and CD86^- T cells and that a neutralizing anti-CD58 mAb (clone TS2/9; 24 poorly affects the capacity of CD86⁺ T cells to activate naive cells (data not shown). These results suggest that the interaction between CD80/CD86 and CD28 could be more potent than the interaction between CD58 and CD2 in providing costimulatory signal to resting naive T cells. Nevertheless, the main point is that CD86 expressed on effector memory T cells is functional and able to costimulate naive T cell responses. To our knowledge, this result is the first demonstration of a functional role of CD86 expressed by human T cells.

In conclusion, this study shows that long term stimulated human T cells express functional CD86. Interestingly, CD86 expression seems to be restricted to effector memory T cells. Moreover, CD86 expressed by T cells can provide the accessory signal required for an efficient priming of naive T cells. As such, although T cells are less efficient than professional APC in presenting Ag, they can express costimulatory molecules that are crucial for the initiation of an immune response. This result suggests that activated memory T cells may favor the proliferation of naive T cells that have been activated via the TCR in a paracrine manner.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Pascale Jeannin, Centre d’Immunologie Pierre Fabre, 5, Avenue Napoléon III, BP 97, F-74164 Saint-Julien en Genevois, France. E-mail address:

² Abbreviations used in this paper: TCC, T cell clones; PFA, paraformaldehyde; L, ligand; PE, phycoerythrin; SI, stimulation index.

Received for publication June 18, 1998. Accepted for publication November 6, 1998.

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