Constitutive intracellular expression and activation-induced cell surface up-regulation of CD44v3 in human T lymphocytes

2.1 Cell surface expression of CD44v3 and CD44v6 on activated human peripheral blood T lymphocytes and T cell lines

Plastic-nonadherent, nylon wool-enriched human pe-ripheral blood lymphocytes were assessed for cell surface expression of the standard CD44 form (CD44H), as well as v3- and v6-containing isoforms by flow cytometry. Cells were counterstained with anti-CD3 antibody to identify T cells. Anti-CD25 (IL-2 receptor) antibody was included as positive control for monitoring T cell activation, and was consistently found up-regulated (Fig. 1).

As it has been reported by several authors 23, 26, 34, 35, unstimulated T lymphocytes expressed CD44H at the cell surface (not shown), while the expression of the variant isoforms was below the limit detectable by flow cytometry or immunofluorescence analysis (Fig. 1, 2A, and 3A). In vitro activation either with anti-CD3 antibody and IL-2, or with the phorbol ester PMA led to the up-regulation of CD44H (not shown), as well as CD44v3- and CD44v6-encoded epitopes (Fig. 1, 2A, and 3B). Confocal microscopy showed that CD44v3 appeared as random receptor aggregates at the surface of CD3⁺ cells (Fig. 3C, D). The intensity of staining for CD44v3 at the surface of T lymphocytes 24 h after activation was in a range similar to the expression level of CD44v6 (Fig. 2A), as well as the percentage of positive cells (∼40-70% after PMA activation, and ∼ 20-50% after OKT3 + IL-2).

The effect of PMA was also studied on the human T cell leukemia lines MOLT-4 and CCRF-CEM. Both splice variants were absent from the surface of unstimulated MOLT-4 cells, while low level of expression was detected on CCRF-CEM cells. PMA treatment resulted in an enhanced CD44v3 and v6 expression on both cell lines (Fig. 2B).

The increased expression of CD44v3 by in vitro activated T cells was transient, showing a time kinetics similar to that reported for CD44v6 26, peaking at 24–48 h (Fig. 4). Similar results were obtained in the case of MOLT-4 and CCRF-CEM lines (data not shown).

Using CD4- and CD8-specific antibodies to identify T cell subpopulations, we demonstrated that the increase in the overall expression of CD44v3 on T lymphocytes resulted predominantly from an up-regulation of this variant on the CD4⁺ subset (Fig. 5).

2.2 Constitutive intracellular expression of CD44v3 in T lymphocytes and T cell lines

To study the intracellular expression of the v3 and v6 exon-encoded epitopes in peripheral blood T cells and T cell subsets, we performed the immune reactions on formaldehyde-fixed and permeabilized cells. Resting T lymphocytes showed intensive intracellular labeling for CD44v3, while they were negative for CD44v6 (Fig. 6A). The intracellular pool of CD44v3 was confined to a tubulovesicular structure in a perinuclear location using confocal microscopy and optical sectioning (Fig. 3E). The analysis of the T cell lines CCRF-CEM and MOLT-4 revealed similar, robust intracellular staining for CD44v3 (Fig. 6B). These results indicate the constitutive expression of v3 exon-containing CD44 variant(s). This was supported by the reverse transcription (RT)-PCR analysis of the two cell lines, showing constitutive mRNA expression of the CD44v3 exon in these cells (Fig. 7A), which was confirmed by the sequence analysis of the PCR product. Beside the ∼ 346-bp major PCR product present in both cell lines (directly spliced v3 exon), a larger product was also detected in CCRF-CEM cells, presumably representing v2-v3 (∼ 475 bp). Western blot analysis of MOLT-4 and CCRF-CEM cell lysates demonstrated a major ∼ 120-kDa and a weaker ∼ 180-kDa band (Fig. 7B).

At the same time, in another time course experiment employing shorter incubation periods, we found that treatment of T cells for 15 min with PMA, a direct activator of protein kinase C, did not induce expression of either CD44 variants studied (not shown). This suggested that de novo protein synthesis may be required for cell surface expression of these variants. To support this assumption, we performed 6-h PMA treatment in the presence of the protein synthesis inhibitor cycloheximide. Fig. 8A shows that the induction of cell surface expression of CD44v3 was blocked by the addition of cycloheximide. The same effect was observed on the up-regulation of CD44v6, while the cell surface expression of the constitutively expressed CD44H was not influenced (not shown). Treatment of T cells with brefeldin A, an agent that has been described as a blocker of protein translocation from the endoplasmic reticulum to Golgi 41, 42, also inhibited PMA-induced cell surface up-regulation of CD44v3 (Fig. 8B).

4.1 Cell separation and culture

Mononuclear cells were separated from peripheral blood of healthy volunteers using Ficoll density gradient centrifugation. Cells were washed in PBS, followed by the removal of adherent cells by incubation in 10% FCS-RPMI 1640 at 37°C in 6-well plastic plates, and T cells were enriched using nylon wool column. The T cell leukemia lines MOLT-4, CCRF-CEM and Jurkat were cultured in RPMI 1640 supplemented with 10% FCS and gentamicin.

For in vitro activation peripheral blood T cells were cultured in 10% FCS-RPMI 1640 either with PMA (10 ng/ml), or in 24-well plates pretreated with OKT3 supernatant (1:400 dilution for 3 h at 37°C, followed by washing three times in PBS), in the presence of IL-2 (100 U/ml; DuPont Med. Prod., Wilmington, DE). CCRF-CEM and MOLT-4 cells were stimulated with PMA (10 ng/ml) for 24 h.

In some experiments T cell stimulation by PMA was carried out in the presence of the protein synthesis inhibitor cycloheximide, or the endoplasmic reticulum to Golgi transport inhibitor brefeldin A 41, 42 (Sigma, St. Louis, MO) at a concentration of 10 μg/ml.

4.2 Antibodies and immunofluorescence

For the detection of cell surface antigen expression, immunofluorescence labeling was performed on unfixed cells at 4°C, or after fixation with 1% buffered paraformaldehyde (PFA). For intracellular antigen detection cells were fixed with PFA for 15 min, followed by permeabilization with 1% saponin for 30 min, and processed at room temperature. Nonspecific antibody binding was blocked using PBS containing 1% BSA. Cells were incubated for 60 min with 10 μg/ml of the primary antibody. Monoclonal anti-CD25 was purchased from DAKO (Glostrup, Denmark); anti-CD44H, anti-CD44v3 and anti-CD44v6 were all from R&D Systems (Abingdon, GB). Murine IgG2b, IgG2a and IgG1 (Sigma) were applied as isotype controls. Cells were washed three times in PBS, then incubated for 60 min with FITC-conjugated goat anti-mouse IgG (1:70; Amersham, Little Chalfont, GB), followed by washing three times in PBS. To identify T lymphocytes or T cell subsets, cells were counterstained with PE-conjugated monoclonal anti-CD3 or anti-CD4 antibodies (Immunotech, Marseille, France) (control: PE-conjugated IgG1, Becton Dickinson, Sunnyvale, CA), or with anti-CD8-Quantum Red (Sigma) for 30 min. Fluorescence was measured by a FACStar flow cytometer (Becton Dickinson).

Surface and cytoplasmic anti-CD44v3-labeled lymphocytes have been also viewed by NIKON Eclipse-E600 epifluorescence microscope (Optoteam, Vienna, Austria) equipped with Spot Junior CCD camera and image analyzing software (Diagnostic Instruments Inc., Sterling Heights, MI). Certain samples were also analyzed by confocal microscopy using LaserSharp software (MRC-1024, Bio-Rad Laboratories GmbH, Munich, Germany). In these cases anti-CD44v3-labeled cells were stained with biotinylated anti-mouse IgG, followed by incubation with streptavidin-FITC (both purchased from Vector Laboratories Inc., Burlingame, CA). Nuclear staining has been performed using propidium iodide (2 μg/ml, 1 min). In the case of double staining, samples were post-labeled with anti-CD3 antibody, followed by incubation with biotinylated anti-mouse IgG and streptavidin-Texas Red (Amersham).

4.3 Western blot analysis

Cells were solubilized in gel sample buffer (5×10⁶/ml), boiled for 5 min, and aliquots of 2.5×10⁵ cell equivalent were separated on 8% SDS-polyacrylamide gels (Mini-Protean II Cell, Bio-Rad Laboratories). Proteins were electrotransferred to PVDF membranes at 200 mA for 130 min, using Mini-Trans-Blot Cell (Bio-Rad Laboratories). Blots were treated with 2% H₂O₂ in 0.1 M Tris pH 7.5/20% methanol for 30 min, then blocked in 10 mM Tris buffer pH 7.9, containing 150 mM NaCl, 0.1% Tween 20, 5% nonfat dried milk, and 2% BSA, for 1 h. Primary antibodies (anti-CD44v3 or isotype control Ig) were applied overnight at 4°C at a concentration of 1 μg/ml diluted in the same blocking buffer. The blots were then incubated in biotinylated anti-mouse IgG (1:1,500, 1 h), followed by incubation with streptavidin-horseradish peroxidase (1:3,000, 1 h) (both from Vector Laboratories). The membranes were washed four times in TBS after each incubation step. Lysate of equal amount of Jurkat cells served as negative control.

4.4 RNA isolation and RT-PCR analysis

Total RNA was isolated from 10⁷ cells using RNAzol^TMB (TEL-TEST, Friendswood, TX), according to the manufacturer's instructions. Of the total RNA 1 μg was used for cDNA synthesis with 0.1 μg oligo(dT)_12–18 (Gibco BRL, Life Technologies, Paisley, Scotland), 1 mM dNTP (Gibco BRL) and 50 U MMLV reverse transcriptase (Promega, Madison, WI) for 30 min at 37°C in a total volume of 20 μl; 2.5 μl of each cDNA products were amplified using CD44 standard region 5´ (AGT CAC AGA CCT GCC CAA TGC CTT T) and exon v3 3´ primer (GGT GTC TGT CTC TTT CAT CTT CAT TTT CTT CAT TT) 55, or 5´ (TTC ACC ACC ATG GAG AAG GCT) and 3´ (ACA GCC TTG GCA GCA CCA GT) primers for GAPDH. PCR amplifications were performed in 25-μl reactions, containing 1.5 mM MgCl₂, 0.25 mM dNTP (Gibco BRL), 1 U Dupl-A-Taq Taq polymerase (Zenon Biotechological Ltd., Szeged, Hungary), and 1 μM of the forward and the corresponding reverse primers. Reactions were performed in a Crocodile III thermocycler (Appligene/Oncor, Illkirch, France) for 35 cycles (94°C for 30 s, 65°C for 30 s, 72°C for 120 s), with final extension at 72°C for 10 min. For negative controls, reactions were performed in the absence of RNA (to exclude contamination in reaction mixture) or in the absence of reverse transcriptase (to exclude potential cDNA contamination of the RNA preparations). For sequence analysis, PCR products amplified with CD44 primers were pretreated with shrimp alkaline phosphatase/exonuclease I (Amersham) according to the manufacturer's instructions. Fluorescent labeling of the pretreated products was carried out using Big Dye^TM Terminator Cycle Sequencing Kit (Perkin-Elmer Biosystems, Foster City, CA). The samples were purified by isopropanol precipitation and run on ABI PRISM 310 Genetic Analyzer (Perkin-Elmer Biosystems).