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(Journal of Leukocyte Biology. 2001;69:215-223.)
© 2001 by Society for Leukocyte Biology

Antibodies recognizing CD24 LAP epitope on human T cells enhance CD28 and IL-2 T cell proliferation

María del C. Salamone, Carolina Rosselot, Gabriela V. Salamone, Marcos Barboza, Miguel Kado and Leonardo Fainboim

Immunogenetic Division, University Hospital, School of Medicine, University of Buenos Aires, Argentina

Correspondence: María del Carmen Salamone, Ph.D., División Immunogenética, Hospital de Clínicas, José de San Martín, Av Córdoba 2351, 3° Piso, (1120) Buenos Aires, Argentina. E-mail: marysasinectis{at}com.ar


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Membrane expression of the CD24 molecule on activated T lymphocytes is not elucidated fully. We previously described the intracellular and cell-surface expression of the CD24 sialic acid-dependent epitope(s) on phytohemagglutinin-activated peripheral blood mononuclear cells. However, the CD24 core protein was not detected previously on human T cells. This study reinvestigated the expression and role of CD24 in T cell subsets. We analyzed binding of anti-CD24 monoclonal antibodies (mAbs) to sialic and leucine-alanine-proline (LAP) epitopes in resting and activated, normal T lymphocytes. CD24 LAP and CD24 sialic epitopes were detected on activated CD4- and CD8-positive cells. Although expression of CD24 sialic epitopes remained stably expressed in interleukin (IL)-2-dependent cultures, T cell expression of the LAP epitope was transient. Anti-LAP antibodies strongly enhanced the response of T cells to a combination of anti-CD3/CD28 mAbs and enhanced proliferative response induced by recombinant IL-2. We found similarities in the tissue distribution and function of the human CD24 LAP molecule and the murine, heat-stable antigen, which suggests that CD24 might function as a signaling molecule on human T cells.

Key Words: CD24 antigen • costimulation signals • PBMC • phytohemagglutinin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The essential first step in the generation of T cell responses is delivered by the interaction of the T cell receptor (TCR) with the major histocompatibility complex (MHC)/peptide complex on the surface of the antigen-presenting cell (APC). Molecules that interact with the TCR are not sufficient to stimulate T cell proliferation or its effector function. Consequently, a two-signal model of lymphocyte activation has been proposed, with the first signal being the TCR-MHC peptide interaction. The second signal is provided by costimulatory molecules CD80 and CD86 on APC, which interact with their receptor(s) CD28 and CD152 on the T lymphocyte membrane [1 , 2 ]. However, there is evidence that other activation mechanisms act independent of the B7/CD28 pathway [3 , 4 ].

The CD24 antigen has an extensive carbohydrate structure attached to a glyosyl-phosphatidylinositol (GPI)-anchored protein core. In the B cell differentiation lineage, this molecule is found from the precursor stage through plasma cells [5 6 7 ]. It also has been detected on mature granulocytes, certain epithelial cells, tumors of neuroectodermal origin, and Sezary cells [8 9 10 11 ]. CD24 molecules have been identified as a set of diverse glycoproteins with apparent molecular weights of 35–45 kD, probably resulting from variation of CD24 glycosylation [12 ]. The structure postulated for CD24 suggests that signaling could be triggered by GPI-anchored, cell-surface molecules complexed to protein tyrosine kinases (PTKs) [13 ]. It has been suggested that the murine, heat-stable antigen (HSA) molecule (homologue to human CD24 antigen) can act as another costimulatory ligand on APC and function as a signaling molecule in T lymphocytes [14 15 16 ]. Moreover, cross-linking CD24 on the surface of B cells by monoclonal antibodies (mAbs) results in a rapid increase in cytoplasmic calcium levels, indicating that CD24 can exert its effect on B cells by initiating a calcium-mobilizing signal transduction pathway [8 ]. It has also been found that this molecule can trigger the respiratory burst, which is an essential part of granulocyte function [10 ]. Some anti-CD24 mAbs can recognize peptidic rather than oligosaccharide epitopes, and blocking experiments have suggested the presence of at least four CD24 epitopes on human B cells [17 ]. The mAbs ML5, OKB2, and SWA11 were found to bind the leucine-alanine-proline (LAP) sequence juxtaposed to the GPI anchorage site [18 ]. We previously showed intracellular and cell-surface expression of CD24 sialic, acid-dependent epitope(s) on phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC) [19 , 20 ]. However, the CD24 core protein was not detected previously on human T cells [21 ]. In the present study, we reinvestigated the cell-surface reactivity of anti-CD24 mAb to LAP and sialic-related epitopes on resting and activated normal T lymphocytes. We found that the LAP epitope was transiently expressed on activated T lymphocytes. Furthermore, antibodies that recognize the LAP epitope could enhance the proliferative response of T cells induced with a combination of immobilized anti-CD3 plus anti-CD28 mAb, or with recombinant interleukin (rIL-2). The results we obtained show not only an important similarity in tissue distribution between murine HSA and human CD24, LAP-positive molecules but also suggest that this antigen could function as a signaling molecule on human T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
Antibodies that recognized CD3, CD4, CD8, CD14, CD19, and CD13 (PE, R-Phycoerythrin conjugate, Caltag, Burlingame, CA) antigens were used for the immunologic phenotyping of the samples. Nonrelevant murine mAb immunoglobulin G (IgG)2a, IgG2a [fluorescein isothiocyanate (FITC)], IgG1, and IgM isotypes were used as negative controls. Unlabeled anti-CD24 mAbs were obtained from the B cell panel of the V International Workshop on Human Leucocyte Differentiation Antigens. 4B10 mAb (anti-CD28) and anti-CD152 (7F8 and 11 D4) mAbs were obtained from the T cell panel of the VI International Workshop on Human Leucocyte Differentiation Antigens. Fluorochome-conjugated ML5 (FITC) mAb was obtained from PharMingen (San Diego, CA). The activation state of the cells was evidenced by their reactivity with anti-CD25 (FITC, Caltag) and anti-HLA-DR (HB55) mAb. Anti-CD3 was purified from UCHT-1 hybridoma supernatants by using protein G affinity chromatography.

Cells
PBMC were obtained from normal donors with confirmed negative serology for hepatitis B virus, human immunodeficiency virus, hepatitis C virus, and Chagas disease. PBMC were separated on a Ficoll-Hypaque gradient. Mononuclear cells were washed and resuspended to 1 x 106/ml cells in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS), gentamicin (100 µg/ml), and fungizone (20 µg/ml). Mitogenic stimulation of PBMC was induced by adding PHA (1 µg/ml) or phorbol myristate acetate (PMA; 10, 15, 20, and 25 ng/ml) and culturing for different lengths of time at 37°C. Additional experiments were also performed in the presence of PMA plus ionomycin (1 µg/ml).

Long-term culture
PHA-activated PBMC from three different donors were cultured as long as 20 days. After 6 days of culture, cells were fed with IMDM medium supplemented with 10% FCS, 100 µg/ml gentamicin, and 20 µg/ml fungizone and were reexposed to PHA (1 µg/ml) and placed into long-term culture in the presence of high concentrations of rIL-2 (80 U/ml). According to the cellular growth detected, this process was repeated every 5 or 6 days. In these experimental conditions, we observed that the IL-2 concentration did not become growth-limiting, assuring the exponential growth and high levels of expression of IL-2 receptor (IL-2R) through the culture.

Immunofluorescence studies
For indirect staining, 106 PBMC cells were washed twice with RPMI 1640/5% FCS preincubated with RPMI 1640/5% AB normal human serum for 30 min at 37°C to exclude nonspecific binding through Fc receptors and incubated with relevant mAbs for 30 min on ice. After washing twice in cold RPMI 1640/5% FCS, cells were stained with FITC goat anti-mouse Ig (GAM/FITC) for 30 min on ice. For two-color immunofluorescence, cells were incubated with blocking Ab to reduce any nonspecific staining by any remaining free F(ab') fraction of GAM/FITC. Then, cells were stained using an additional fluorochrome-conjugated mAb (anti-CD3-PE, anti-CD4-PE, or anti-CD8-PE).

For double-direct staining, 106 PBMC were incubated with fluorochrome-conjugated mAbs for 15 min at room temperature.

After staining, cells were washed in phosphate-buffered saline (PBS), and antigen expressions were measured by flow cytometry using a Facstar plus analyzer (Becton Dickinson, CA) or an Ortho CytoronAbsolute (Raritan, NJ). A propidium iodide exclusion gate was pre-set to ensure that only viable cells were acquired. Analysis was performed on the lymphocyte fraction, excluding monocytes and polymorphic cells. The regions were gated based on forward and scatter parameters. In all experiments, background threshold levels were set using irrelevant mouse Ig and an additional fluorochrome-conjugated mAb (anti-CD3-PE, anti-CD4-PE, or anti-CD8-PE). These controls allowed us to establish the optimal cutoff channels for Y and X axis for each population analyzed.

Cell proliferation assay
PBMC were activated at 3.5 x 104 cells/well in 96-well microtiter plates using a combination of anti-CD3 (UCHT-1) and anti-CD28 mAb (4B10). Wells were coated previously with anti-CD3 in buffer carbonate (pH 9.6). For optimal CD3-cross-linking (the 3H thymidine incorporation was >=60,000 cpm), wells were incubated with 30 µl of the anti-CD3 mAb at a final concentration of 5 µg/ml, whereas for suboptimal activation (the 3H thymidine incorporation was <=10,000 cpm), the coating was applied with 30 µl of a final concentration of 2 µg/ml. The optimal concentration of ascites fluids of anti-CD28 and anti-CD24 mAb was established by titration, and finally, it was added at a 1/200 final dilution. Cells were cultured for 3 days, [3H] thymidine (1 µCi/well) was added for an additional 18 h, and cells were then harvested. Additionally, PBMC suspended in IMDM medium supplemented with 10% AB normal human serum were stimulated with different concentrations of rIL-2 (at 3.5x104 cells/well/100 µl in 96-well microtiter plates) plus 1/200 final dilution of anti-CD24. After 54 h of stimulation, 1 µCi/well [3H] thymidine was added, and the proliferation was determined on day 3 of culture. 3H-thymidine incorporation was measured with a beta liquid scintillation counter. Results are expressed as the mean of triplicate counts per minute (cpm); the standard error of the mean of triplicate wells was usually less than 10%. Statistical analysis was done using the Bonferroni multiple comparisons test.

Enriched T cell fraction
PBMC were first depleted of adherent cells by incubation on plastic Petri dishes. After 2 h of culture in RPMI-1640 medium at 37°C, nonadherent cells were removed, and T cells were further enriched by two different treatments. The nonadherent fraction was incubated with an anti-CD20 mAb (93-1B3) followed by incubation with anti-mouse, Ig-coated magnetic beads. In another sets of experiments, T cells were enriched by negative selection with magnetic beads coated with anti-HLA-DR mAb to remove B cells. The purity of the T cell-enriched fraction, verified by fluorescein-activated cell sorter (FACS) analysis, showed a contamination of 1%–3% of monocytes and 0.5%–1.5% of B cells. To obtain these results, two cycles of negative selection were eventually performed.

The index of enhancement (IE) was calculated as: % IE = (cpm of activator + anti-CD24 mAb) - (cpm of activator) x 100/(cpm of activator).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Membrane reactivity of CD24 mAb on resting and activated CD4+ and CD8+ T cells
The expression of CD24 on human T cells is not fully elucidated. Previously, we showed intracellular and cell-surface expression of CD24 sialic, acid-dependent epitope(s) on PHA-activated PBMC [19 , 20 ]. However, the CD24 core protein was not detected on human T cells previously [21 ]. We reinvestigated this question by studying the expression of CD24 epitopes on resting and activated CD4+ and CD8+ T cell subsets. Table 1 shows the range of reactivity of anti-CD24 antibodies tested on resting PBMC from 10 healthy donors. Their blood samples were tested by double immunofluorescence and evaluated by flow cytometric analysis. According to a previous study [21 ], antibodies that recognized the sialic acid epitope (Table 1 , Group I) reacted with CD4+ (range of expression, 66–99%) and CD8+ T cells (range, 18–86%). Antibodies SWA11, ML5, and OKB2, which recognize the linear protein epitope LAP (Table 1 , Group II), showed poor reactivity to T cell subsets (ranges, 0–4% and 1–15% for CD4+ and CD8+ cells, respectively). We next evaluated the reactivity of anti-CD24 mAb on activated, mature PBMC. Expression of this sialoglycoprotein was regulated by and dependent on PHA stimulation. Kinetic studies carried out after 0, 2, 3, and 6 days of culture (n=5) indicated that anti-CD24 antibody reactivity increased on CD3+ cells in a time-dependent manner. Figure 1a shows a representative experiment of the reactivity of the previously defined groups (Groups I and II) of antibodies on activated PBMC. Mitogenic stimulation up-regulated the mean fluorescence intensity (MFI) of surface CD24 LAP and sialic-related epitope expression on both T cell subsets (compared with nonstimulated T cells and the respective negative controls). The MFI on CD3+ cells was always lower than that observed on non-T cells. The sialic epitope group, which was clearly expressed on CD4+ and CD8+ cells, showed a maximal MFI between 3 and 6 days after PHA activation (Fig. 1a) . The LAP epitope was also detected on activated CD4+ and CD8+ T cells but with a percentage and an MFI lower than that observed with antibodies that recognized sialic epitopes. The reactivity of all anti-CD24 mAbs showed discrepancies among different donors, with a wide range of positive reactivity. However, these differences were more evident with antibodies recognizing LAP. The peak expression of LAP was usually detected 2 days after mitogenic activation and decreased after that. The maximum expression on CD4+ and CD8+ cells observed at 48 h was 25–58% and 19–50%, respectively. However, some donors showed peak expression of LAP at 6 days after activation (Fig. 1a) . Figure 1b shows two donors, one with peak expression at 48 h (Donor 1) and the other at 144 h (Donor 2). In both cases, this expression is transient, with rapid down-modulation from the cell surface after peak expression.


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  		Table 1. Reactivity of Anti-CD24 mAb on Resting T Cells

 


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  		Figure 1. Kinetic expression of CD24 epitopes on resting and activated peripheral blood CD4+ and CD8+ cells. (a) PBMC were cultured for different lengths of time in IMDM medium supplemented with 10% of FCS and 1µg/ml PHA. After 6 days of culture, cells were fed with fresh medium and restimulated with 1 µg/ml PHA and 80 U/ml IL-2 (final concentration). CD24 antigen was stained by indirect immunofluorescence (horizontal axis), and T cell subsets were labeled with an anti-CD4 (PE) or anti-CD8 (PE; vertical axis; see Materials and Methods). Data are shown as two-color, dot-plot charts, with the reactivity of BA-1 and SWA11 mAb recognizing the sialic and LAP epitopes, respectively. On activated T cells, LAP epitope expression was clearly detected with the ML5 and SWA11 mAb. Dot-plot charts of cells stained with anti-CD4 (PE) or anti-CD8 (PE) versus an irrelevant IgG2a mAb used as negative control were also included. The horizontal and vertical axes show the logarithmic MFI. The MFI detected on CD4- and CD8-positive cells was lower than that detected on non-T cells ({blacktriangledown}). CD24 showed a down-modulation at day 7 (24 h after IL-2/PHA addition). Whereas the sialic epitope was up-regulated at day 9, there was no reexpression of LAP epitope. The percentage of positive cells, shown in the upper right quadrant, represents the percentage of binding on CD4- and CD8-gated cells. (b) Expression of LAP epitope in two different donors, showing two different kinetics. The horizontal axis indicates time after PHA activation. The vertical axis shows the percentage of binding on gated cells (CD4+ or CD8+). (c) Counter-plot chart shows the expression of the sialic epitope on CD3-positive cells after 20 days of culture. The percentage of positive cells depicted in the upper right quadrant represents the percentage of binding on CD3-gated cells (region R1).

 
The expression of CD25 and HLA-DR antigens on CD3+ cells was used as a positive control of activation. The expression at 48 h increased to values of 50–66% for CD25 and 57–70% for HLA-DR. After 6 days of culture, their expression was 72–88% and 68–86% for CD25 and HLA-DR, respectively (n=5; not shown).

The expression of CD24 epitopes was also evaluated after 20 days of culture. IL-2 receptor density determines the proliferation of cells in the proliferative phase of the cell cycle. When lectin-activated T cells acquire IL-2 receptors, the maintenance of continued proliferation depends on adequate concentrations of IL-2 [22 ]. To maintain exponential growth, expression of high levels of IL-2 receptor and nonlimiting concentration of IL-2 are necessary. For this purpose, cells were re-exposed to PHA every 5 or 6 days (1 µg/ml) and cultured in the presence of 80 U/ml of rIL-2. Under these conditions, CD25 and HLA-DR remained stably expressed during the 20 days of culture in more than 90% of cells. Similar to sialic epitope expression, its MFI increased until day 6 and was down-modulated on day 7. This decrease in the expression of the sialic epitope followed the addition of rIL-2/PHA (or rIL-2 alone; not shown) on day 6 (see Fig.1a , day 7). As also depicted in Figure 1a at day 9, the CD24 sialic epitope was clearly up-modulated. At the end of long-term culture (day 20), the sialic epitope was expressed by more than 80% of CD3+ cells (Fig. 1c) . These results were confirmed in three independent experiments.

The expression of CD24 LAP epitope was also analyzed in long-term culture. As described previously, the kinetics of the LAP epitope varied among different donors. Only a few individuals showed a peak LAP expression at day 6 of activation. Among those individuals, the addition of the rIL-2/PHA at day 6 was accompanied by a decrease in LAP expression (Fig. 1a , day 7). Several days later, it was evident that the T cells did not maintain the capacity to up-modulate this epitope at levels similar to those detected in the early periods of culture (Fig. 1a , day 9). LAP was not reexpressed after 20 days of culture in the same conditions as was found for the up-regulation of the sialic epitope (<=12% on CD3+ cells; not shown). We also studied a donor who showed maximal LAP expression 2 days after activation. FACS analysis of the cells grown for 20 days showed that expression of LAP epitope was not re-established after that.

LAP was also expressed when PBMC were stimulated with PMA (Fig. 2a ). After 48 h of PMA activation, LAP was present in 28–33% of gated CD3+ cells (Fig. 2a 2b) and again decreased at 72 h. Figure 2b also shows the kinetics of expression of CD25 and HLA-DR, as indicators of activation in response to PMA. Conversely, the addition of ionomycin to PMA stimulated PBMC; although it increased the MFI of CD25, it did not affect the expression of LAP (Fig. 2c) .



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  		Figure 2. Expression of CD24 protein core on activated PMA CD3+ cells. (a) PBMC were cultured in IMDM medium supplemented with 10% FCS and 10 ng/ml PMA. After 2 days of culture, cells were stained by double-direct immunofluorescence with the anti-CD24 ML5 (FITC) mAb (horizontal axis), and the T cell fraction was identified by labeling with an anti-CD3 (PE) mAb (vertical axis; see Materials and Methods). Data are shown as two-color, dot-plot charts; the results are representative of three separate experiments. A dot-plot chart of cells stained with anti-CD3 (PE) versus an irrelevant IgG2a (FITC) mAb used as negative control was also included. The percentage of cells detected in each quadrant is shown in the upper right quadrant. (b) Reactivity of ML5 (FITC) on resting and activated PMA CD3+ cells. Cells were cultured in the presence of 10 ng/ml PMA for different lengths of time. The reactivity of ML5 (FITC), anti-CD25 (FITC), and HLA-DR was analyzed on CD3+ gated cells (filled histograms, region R2 in panel a). The histograms of cells stained with the negative control mAb, gated on CD3+ region (region R2 in panel a), were also overlapped (outlined histograms). ML5 (FITC) showed a poor reactivity on resting CD3+ cells and after 24 h of activation. The reactivity was clearly increased after 2 days of culture, after which it decreased. The reactivity with anti-CD25 and anti-HLA-DR clearly shows a high activation state of the cells. The results are representative of two separate experiments. (c) Reactivity of ML5 (FITC) and CD25 (FITC) on CD3+ gated cells, activated for 48 h with 10 ng/ml PMA (filled histograms) and 25 ng/ml PMA plus 1 µg/ml ionomycin (gray-outlined histograms). The negative controls were also included (n.c. histograms). Additional samples, incubated only with 15, 20, or 25 ng/ml PMA, showed a similar level of ML5 reactivity (not shown).

 
Functional effects of anti-CD24 mAb on PBMC and T cell proliferation
Having established the expression of CD24-LAP epitope on activated T lymphocytes, we studied the effect of antibodies recognizing this epitope on PBMC proliferation. As shown in Figure 3a , the addition of anti-LAP mAb (SWA11) to PBMC that were activated by suboptimal quantities of anti-CD3 (2 µg/ml) plus anti-CD28 mAb showed strongly enhanced cell proliferation. Three independent experiments showed an index of enhancement that ranged between 46% and 76% (p<0.001). This effect decreased with increased anti-CD3 concentration, reaching {cong}22% when PBMC were stimulated by optimal concentrations of anti-CD3 (5 µg/ml, Fig. 3b ). The combination of coated anti-CD3 and soluble anti-CD24 mAb showed a higher proliferative response than the combination of coated anti-CD3 plus irrelevant mAb, but this difference was not statistically significant (Fig. 3a , n=3, p>0.05). Anti-CD152 mAb, 11D4, or 7F8, used with anti-CD3 alone or in combination with anti-CD3 plus anti-CD24 (SWA11), did not induce any significant effect (Fig. 3a and 3b) . Similarly, anti-CD24 alone or together with anti-CD28 induced a nonstatistically significant effect on the proliferation of PBMC (not shown). These results indicated that SWA11 was not stimulatory by itself but could enhance a proliferative response induced by anti-CD3 plus CD28. To determine whether this cell expansion was a result of B lymphocyte proliferation, PBMC depleted of adherent cells and B cells were used as the source of a T cell-enriched population. When the proliferation assays were performed, the combination of anti-CD3/CD28/CD24 (LAP) mAbs also induced an enhancement in this cell fraction compared with the expansion detected using anti-CD3 plus anti-CD28 mAb alone, indicating that the expansion detected on PBMC was because of, at least partially, T cell proliferation (Fig. 3c) .



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  		Figure 3. Effect of anti-CD24 mAb on CD3 plus CD28 activation pathway. (a) PBMC were activated by immobilized anti-CD3 mAb (suboptimal concentration, 2 µg/ml) plus anti-CD28 (CD3/CD28). Three different experiments showed that the addition of anti-LAP mAb (SWA11) increased the proliferative response induced by anti-CD3/CD28 mAb [n=3, CD3/CD28/CD24 (LAP), p<0.001]. The addition of anti-CD152 mAb (11D4 or 7F8) to anti-CD3 (CD3/CD152) or to a combination of anti-CD3 plus anti-CD24 [CD3/CD152/CD24 (LAP)] was not significant. Anti-CD24, anti-CD28, and anti-CD152 mAbs were added at a final concentration of 1/200. Representative results from one of three separate experiments are shown. The graphics show the mean ± standard error of the mean (SE) obtained from triplicate data. (b) PBMC activated by coated anti-CD3 mAb (optimal concentration, 5 µg/ml) and costimulated by anti-CD28 and anti-CD24 mAbs [n=2, CD3/CD28/CD24 (LAP), p<0.05]. CD24 (LAP) and CD24 (sialic) indicate the addition of SWA11 and HB9 mAbs, which recognized the LAP and sialic epitopes, respectively. Representative results from one of two separate experiments are shown. The graphics show the mean ± SE obtained from triplicate data. (c) Costimulatory effect detected on an enriched T cell fraction [obtained by incubation of nonadherent cell fraction with an anti-CD20 mAb (93-1B3) followed by incubation with anti-mouse Ig-coated magnetic beads; see Materials and Methods] activated by suboptimal concentration of anti-CD3 mAb (2 µg/ml). Representative results from one of two separate experiments are shown. The graphics show the mean ± SE obtained from triplicate data [n=2, CD3/CD28/CD24 (LAP), p<0.05]. Proliferation of cells was determined at day 4 of culture. In a–c, the vertical axis shows the mAb tested, and the horizontal axis shows the 3H thymidine incorporated in cpm (d, e, f). Stimulatory effect of anti-CD24 mAb on the IL-2 activation pathway. (d) Anti-LAP mAb enhanced the proliferative response of PBMC stimulated with 20 U/ml rIL-2. (ML5/IL-2, p<0.05, and SWA11/IL-2, p<0.001). Additionally HB9 mAb, which recognizes the sialic epitope, also showed a statistically significant enhancing effect (p<0.05). The horizontal axis shows the different mAbs tested, and the vertical axis shows the 3H thymidine incorporated in cpm. The graphics show the mean ± SE of six different experiments. (e) Stimulatory effect of anti-CD24 mAb on the proliferation of an enriched T cell fraction (obtained by incubation of nonadherent fraction with anti-HLA-DR-coated magnetic beads; see Materials and Methods) cultured in the presence of 20 U/ml IL-2. The graphics show the mean ± SE obtained from two different experiments (IL-2/SWA11, p<0.001, IL-2/ML5, p<0.05). The horizontal axis shows the different mAbs tested, and the vertical axis shows the 3H thymidine incorporated in cpm. (f) Index of enhancement detected by addition of SWA11 and ML5 mAb on T cell activated by 0, 20, 60, and 100 U/ml rIL-2. Similar results were obtained from two unrelated donors. The vertical axis shows the % IE, and the horizontal axis shows different concentrations of IL-2 tested. Proliferation of cells was determined after 72 h. NC, negative control.

 
Signaling through CD28 is known to augment IL-2 production via increased IL-2 transcription and mRNA stabilization. Thus, we investigated whether the anti-LAP effect was related to the IL-2 activation pathway. As described previously, the additive effect on T cell proliferation induced by anti-CD3 plus anti-CD28 mAbs was demonstrated using suboptimal concentrations of anti-CD3. We also speculated that using a low concentration of IL-2 might help us investigate the role of LAP in this activation pathway. The proliferative response of PBMC stimulated with 20 U/ml rIL-2 was enhanced significantly by addition of SWA11 and ML5 (Fig. 3d) . The same enhancement effect was observed on enriched T cells from two different donors. As shown in Figure 3e , the addition of SWA11 or ML5 anti-LAP antibodies in the presence of 20 U/ml rIL-2 induced indices of enhancement of 37% and 85%, respectively. The costimulatory effect appeared to be dependent on rIL-2 concentration and varied among donors. The maximal index of enhancement was obtained when between 20 and 60 U/ml rIL-2 was used. Some donors showed the maximal enhancement effect when cells were stimulated with 60 U/ml of rIL-2 (Fig. 3f) . At a concentration of 100 U/ml rIL-2, the enhancement effect was no longer observed.

The effect of the HB9 mAb, which recognizes the sialic epitope, was also analyzed. A stimulatory effect was detected when this antibody was added to PBMC stimulated with 20 U/ml rIL-2 (Fig. 3d) ; however, this effect was not statistically significant when purified T cells were used instead of PBMC (not shown). No enhancement effect was observed when HB9 mAb was added to PBMC (Fig. 3b) or enriched T cells (Fig. 3c) activated by anti-CD3 plus anti-CD28.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human CD24, a cell-surface molecule expressed with a GPI anchor, is considered a differentiation marker for B lymphocytes, but it has also been found on granulocytes, monocytes, neoplasic cells, epithelial cells, and Langerhans cells [5 6 7 8 9 10 ]. In previous studies, we investigated the intracellular and cell-surface expression of CD24 sialic acid-dependent epitope(s) on PHA-activated PBMC [19 , 20 ]. The present study not only confirmed our previous findings but also demonstrated that the LAP epitope is expressed transiently on activated T lymphocytes. This expression was observed on PHA- and PMA-activated T cells, seemed to be highly regulated, and remained relatively constant even in the presence of additional stimulus. To determine the biologic relevance of our results, we explored the effect of antibodies that recognize the CD24 LAP epitope on activated T cells. We found that they can enhance T cell proliferation. It is well known that signaling through CD28 on the surface of T cells promotes the synthesis of IL-2. Our results indicated that the CD24 LAP epitope might function as an activation, signal-transducing molecule that also involves the IL-2 pathway. This biologic function also seemed to be partially regulated by the activation status of the cells, because it became evident only with suboptimal stimulus. Our functional studies suggested that CD24 LAP epitope expression also might be induced after CD28 or IL-2 T cell activation. In line with that was the observation that the LAP epitope was detected on CD3+ cells that were activated only with rIL-2 in the absence of additional signals originating from the TCR (unpublished results).

Our results also indicated that CD24 LAP-positive molecules might be related to the murine CD24 HSA. Genetic analysis indicated that murine and human CD24 evolved from the same ancestral gene [23 ], but their core proteins diverged during evolution [24 ]. Several studies suggested that the HSA might act as a CD28-independent costimulation molecule responsible for clonal expansion and functional differentiation of T cell subsets [14 , 15 , 25 26 27 28 29 ]. Liu et al. [14 ] demonstrated that HSA expressed on lipopolysaccharide (LPS)-activated B cells also functioned as a costimulatory molecule associated with the development of CD4+ cells. In addition, studies performed in knockout mice suggested that CD28 and HSA can act as costimulatory molecules involved in two different activation pathways [25 ]. Whether CD28 and CD24 LAP-positive molecules in humans function as a redundant costimulatory pathway or in an independent manner remains to be elucidated.

In this study we also found that CD24 LAP functions as an activation marker, with transient expression on the membrane of the activated T lymphocytes . In contrast to CD24 antibodies, which recognize carbohydrate epitopes that react with resting and activated T cells, anti-LAP mAbs showed only transient expression on activated T lymphocytes. The transient expression of the LAP epitope detected in this study might explain why it was not previously detected on activated T lymphocytes. Based on the lack of expression of the LAP epitope and the positive reactivity of the sialic-related epitope mAb on resting and activated T cells, Williams et al. [21 , 30 ] defined a molecule called the CD24 carbohydrate-related epitope (CD24cre). Our findings and those of Williams et al. led us to postulate that at least two different molecules exist on the surface of human T lymphocytes, one defined as CD24cre (or LAP negative) [21 ] and the other described here as CD24 LAP positive. Apparently, their simultaneous expression would be extremely variable and would occur only during a very short period, i.e., between 24 and 48 h after activation (Fig. 1b , Donor 1). This transient expression might explain why mRNA was not detected previously on activated T cells.

Studies on human cell lines showed that the CD24 gene has a 5' flanking region with cell type-specific promoter activity. On the adenocarcinoma cell line A549, very low amounts of CD24 message were associated with a considerable level of CD24 core protein expression. These results suggested that post-transcriptional control of CD24 expression might also occur [31 ]. According to our results, we speculate that the CD24 gene might display rapid and transient expression on T cells, followed by rapid degradation at the RNA level, as shown for other early genes [32 , 33 ]. The CD24 transcript might have a kinetic that peaks earlier than when maximal protein expression was usually observed on activated T cells. This tight regulation could ensure transient activation of this gene and contribute to prevention of uncontrolled expression of CD24 core protein implicated in lymphocyte proliferation. The present study provided evidence that CD24 LAP positive molecules expressed on activated human T cells were involved in delivering costimulatory signals, as described for murine HSA CD24 [16 ]. This expression might be regulated strictly to prevent uncontrolled T cell expansion. The role of the costimulatory molecules in regulating T cell responses throughout the course of an autoimmune disease is well known [34 35 36 37 ]. Although data on humans are limited, the finding that CD24 LAP epitope expression is greatly regulated on activated T cells and the similarities with murine HSA lead us to speculate that this molecule might have a physiologic function in vivo in autoimmune disease, or it could be involved in peripheral tolerance. A recent study showed an essential function of HSA in the development of experimental autoimmune encephalomyelitis (EAE). It was shown that expression of HSA on T cells and non-T cells determined the pathogenicity of self-reactive T cells [38 ]. Targeted mutation of HSA abrogated the development of EAE, and soluble HSA drastically ameliorated EAE after autoreactive T cells had been primed. Our results showed that although CD24cre expression remained relatively stable throughout the different T cell states, CD24 LAP expression was transient and greatly regulated. Signaling through the LAP epitope, but not through the sialic epitope, might be involved in T cell expansion, suggesting that uncontrolled expression of CD24 LAP+ molecules might result in uncontrolled T cell clonal expansion. This hypothesis is supported by the finding that CD24 has an associated PTK activity (p56), which is required for signal transduction [13 ]. Similar to our results in humans, the addition of anti-HSA mAb together with anti-CD3 plus anti-CD28 mAbs to previously activated mouse T lymphocytes enhanced the proliferative response significantly [16 ].

Finally, it is known that CD28 provided a necessary costimulus for initiation of T cell responses [1 , 2 ], but not all T cell-mediated immune responses were CD28-dependent. In this context, we should remember that in humans, about 50% of CD8+ T cells are CD28-dependent, indicating that some immune responses can involve additional costimulatory pathways [3 ]. Alternative costimulatory molecules appear to act at different stages of T cell activation and differentiation or to promote the development of a different effector function. Understanding the cellular regulatory pathway that determines CD24 LAP epitope expression on T cells would be important for the design and implementation of tolerance-inducing strategies.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from University of Buenos Aires (TM16, JM21) and CONICET (PEI N0 0185/98). M. d. C. S. and L. F. are members of the scientific career of CONICET. G. V. S. is a fellow of CONICET.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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