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* Departamento de Bioquímica y Biología Molecular (B) e Inmunología, Facultad de Medicina, Campus de Espinardo, Murcia, Spain; and
Servicio de Inmunología, Hospital Universitario Virgen de la Arrixaca, Murcia, Spain
1 Correspondence: Departamento de Bioquímica y Biología Molecular (B) e Inmunología, Facultad de Medicina, Campus de Espinardo, Murcia 30100, Spain. E-mail: trini{at}um.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: T lymphocytes • cytotoxicity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CD33 (siglec-3), the smallest member of the family, was cloned several years ago [6 , 7 ]. It contains two Ig-like extracellular domains and two ITIM-like sequences in their cytoplasmic domain [1 , 8 , 9 ]. After phosphorylation, CD33 is capable of recruiting the protein tyrosine phosphatases Src homology-2-containing tyrosine phosphatase-1 (SHP-1) and SHP-2 [10 , 11 ] and may function as an inhibitory receptor by coligation with CD64 [10 ] on myeloid cells. Anti-CD33 monoclonal antibodies (mAb) prevent the generation of dendritic cells (DC) from monocytes and myeloid CD34+ precursors [12 ] by inducing apoptosis [12 , 13 ]. More recently, a role of protein kinase C in the serine phosphorylation of CD33 and its effect on lectin activity have been described on myeloid cells [14 ]. However, it is not known whether CD33 functions as an inhibitory receptor, and if so, what are the ligand receptor systems within their cellular targets.
CD33 expression is assumed to be restricted to the myeloid lineage of immune cells [1 ]. Thus, it is highly expressed on myeloid-committed cells of the bone marrow and circulating monocytes. CD33 expression is down-regulated to low levels on peripheral granulocytes and resident macrophages, and it is constitutive on DC [15 ]. This expression pattern suggests a role of CD33 on myeloid differentiation and cellular function of monocyte and DC. Although some authors have reported its expression on certain lymphoid cells, this subject has not been completely established up to date. Thus, a low CD33 surface expression was found by flow cytometry on the earliest precursors of fetal thymocytes and a small subset of the postnatal CD34+ human thymocytes [16 ]. Some CD33-expressing T lymphocytes were also detected by flow cytometry, such as CD4+ T lymphocytes immortalized by human T cell leukemia virus-I infection [17 ], some human T cell clones (TCC) stimulated with anti-CD3 mAb plus interleukin (IL)-2 [18 ], or some polyclonal T cells stimulated with anti-CD3 mAb plus different cytokines [19 ]. With the same approach, a subset of CD33+ natural killer (NK) cells could be found in the human umbilical cord [20 ], on some B cell precursors [21], and a subset of CD19+CD33+ B cells in patients with Behcet’s disease and sepsis [22]. A percentage of lymphoproliferative leukemia cells also stained with anti-CD33 mAb is considered an aberrant expression of this antigen [23 ]. In the present work, we have established the expression of CD33 on lymphoid cells by several approaches. We found that activated T and NK cells express the CD33 protein, displaying lower molecular weight than myeloid CD33. Our studies showed that triggering CD33 partially inhibited effector function on reverse antibody-dependent cell-mediated cytotoxicity (rADCC) and natural cytotoxicity mediated by NKL cells, although this effect was not observed on the redirected CD3-mediated cytotoxicity by T cells. Moreover, two isoforms of CD33, generated by alternative splicing, could be expressed in all CD33-positive cells. Both isoforms could play a role in the regulation of myeloid and lymphoid immune function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used the following mAb in the rADCC assays: UCHT1 (anti-CD3, eBioscience, San Diego, CA); KD1 (anti-CD16), HP-F1 [anti-Ig-like transcript 2 (ILT2)], and HP-3B1 (anti-CD94) mAb (a generous gift from Dr. Miguel López-Botet, Universitat Pompeu Fabra, Barcelona, Spain [24 ]).
Cells, cell lines, and TCC
Human myeloid cell lines U-937 and HL-60, the Epstein-Barr virus-transformed B cells GUS and LG-2, the Ramos Burkitt lymphoma, the NK cell line NKL (kindly provided by Dr. Michael. Robertson, Indiana University Medical Center, Indianapolis, IN), the K562 erytroblastoma, the P815 mouse mastocytoma cells, and the 293T human epithelial cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS [tissue-culture medium (TCM)]. The T cell acute lymphoblastic leukemia (T-ALL) 103/2 
T cell line was kindly provided by Dr. Daniela Santoli (The Wistar Institute, Philadelphia, PA). Human peripheral blood cells (PBLs) were obtained from healthy volunteers and purified by discontinuous density gradient in Lymphoprep (Nycomed Pharma, Oslo, Norway) according to standard protocols. The polyclonal T cell lines used in this work were generated by stimulation of fresh PBLs with PHA, anti-CD3 mAb, or coculturing with allogenic lymphoblastoid cell line (LCL)-GUS and allogenic human splenic cells. TCC were obtained by limiting dilution from 10-day-stimulated PBLs as described previously [25
]. After 3–4 weeks from the last stimulation, all T cells were restimulated for further expansion by coculture with irradiated human allogenic mononuclear splenocytes plus irradiated LCL-GUS and rIL-2 (50–100 U/ml) as described [25
]. After 1 week of culture under stimulation conditions, all T cells were maintained in TCM supplemented with rIL-2 at 50–100 U/ml.
Immunofluorescence analysis
Phenotypic analysis of cells was carried out by immunofluorescence on a FACSCalibur cytometer (Becton Dickinson) after staining with the appropriated combination of fluorochrome-conjugated mAb, as described previously [26
]. Cytofluorometer data were analyzed by using CellQuest and Paint-a-Gate programs, all from Becton Dickinson. A minimum of 4000 events per sample was analyzed.
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA from cultured cells was obtained by using the Micro RNA isolation kit (Stratagene, La Jolla, CA) according to the protocol provided by the manufacturer. cDNA was synthesized from 1 to 2 µg RNA using oligo(dT)12–18 and the SuperScript II RNase H RT, according to the protocol of Life Technologies. Based on the human CD33 cDNA sequence (GenBank XM-057602), the following forward (containing the ATG initiation codon) and reverse (designed in the 3'-untranslated region) primers were used to amplify, by RT-PCR, the complete open reading frame (ORF) of CD33: CCT CAG ACA TGC CGC TGC TG and CAG AGA AGA AAA TGG AGA CAT GG. PCRs were run with an annealing temperature of 66°C, 1.5 mM Mg2+, and Ecotaq DNA polymerase (5 U/ml, Ecogen, Barcelona, Spain). The above pair of primers amplified two fragments of 1385 and 976 base pairs (bp), CD33M and CD33m, respectively. The PCR performed with the forward primer CAG GGG CCC TGG CTA TGG and the reverse primer CTG TCA CAT GGA CAG AGA GC amplified the exon 2 of CD33. The exon 2 fragment (373 bp) was used as a probe in Northern blots to detect CD33M transcripts.
Northern blot analysis
Total RNA from different cell lines was obtained as described above. Equal amounts of RNA (20 µg per sample) were denatured for 1 h at 55°C with glioxal and dimethyl sulfoxide and electrophoresed on 1% agarose gels in 10 mM sodium phosphate buffer, pH 7.0. Acridine orange staining of the gel before blotting ascertained that the amounts of RNA loaded were equivalent in all lanes and determined the position of the 28 s and 18 s RNA fractions. Transfer to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ), prehybridization and hybridization, was performed according to Martínez-Esparza et al. [27
]. To identify the CD33 transcripts, two different probes were used: the 976-bp PCR-amplified CD33 fragment (CD33m) and the 373-bp PCR fragment corresponding to CD33 exon 2. The probes were labeled by random priming in the presence of 
-32P deoxy-cytidine 5'-triphosphate by using the Ready-to-Go T-primed first-strand kit from Amersham Pharmacia Biotech (Piscataway, NJ). The 450-bp PCR fragment amplified from glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA, using a commercial set of primers (Clontech, Heidelberg, Germany), was finally used as probe for normalization of phosphorimaging data. Hybridization was performed at 42°C in 50% formamide (vol/vol), 5x saline sodium citrate (SSC; 150 mM NaCl, 15 mM trisodium citrate), 5x Denhardt’s solution, 0.5% (wt/vol) sodium dodecyl sulfate (SDS), 0.2 mg/ml bovine serum albumin, 100 µg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate. Afterwards, the filters were washed twice for 10 min at room temperature in 2x SSC and 0.1% SDS and then washed twice at 55°C for 10 min in 0.1% SSC and 0.1% SDS. The radioactivity associated to each band was quantified in a BioRad GS-525 molecular imager. Filters were then exposed for appropriate times at –70°C with Hyperfilm MP autoradiography film (Amersham Pharmacia Biotech) and Siemens intensifying screens. Each filter was stripped and rehybridized to test all three probes.
Cell-surface labeling and IP
Cell-surface proteins were labeled by biotinylation according to the protocol of the cellular labeling kit used (Boehringer Mannhein, Germany). Briefly, cells were washed twice in phosphate-buffered saline (PBS), pelleted, and resuspended in borate buffer at 107 cells/ml. Biotinylation reagent was added at 5 µg/ml and incubated under agitation at room temperature in the dark. After 15 min of incubation, NH4Cl (50 mM, final concentration) was added to stop the reaction and block free biotin. Then cells were washed twice in cold PBS and prepared for pervanadate stimulation or lysis and IP. Nonstimulated cells were resuspended in lysis buffer [50 mM Tris/ClH, pH 7.5, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride (PMSF)] containing 2 µg/ml protease inhibitors (leupeptin, aprotinin, chemostatin, pepstatin). After 20 min on ice, lysates were centrifuged at 12,500 rpm for 15 min, precleared twice with protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech, Uppsala, Sweden), and immunoprecipitated overnight with protein A-Sepharose CL-4B beads, which had been preincubated previously with anti-CD33 mAb (Serotec, Clone WM53). Subsequently, immunoprecipitates were washed five times with lysis buffer, resuspended in electrophoresis sample buffer, boiled at 95°C for 5 min, and separated by SDS-polyacrylamide gel electrophoresis (PAGE). Finally, isolated immunoprecipitates were blotted onto polyvinylidene difluoride membranes, probed with horseradish peroxidase (HRP)-conjugated streptavidin, and developed using the enhanced chemiluminiscence (ECL) method (Amersham Pharmacia Biotech).
Pervanadate stimulation assays
Biotinylated cells (107) were incubated for 5 min at 37°C in 1 ml RPMI alone or with 0.5–0.1 mM sodium pervanadate, which inhibits phosphatase activity and thus, increases protein tyrosine phosphorylation. Cells were then lysed at 4°C in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.1 mM sodium orthovanadate, 10 mM NaF, 1% NP-40, 1 mM PMSF, 2 µg/ml protease inhibitors, leupeptin, aprotinin, chemostatin, and pepstatin). Lysates were immunoprecipitated with mouse IgG1, anti-CD33 (WM53), or anti-ILT2 (HP-F1) as described above. Precipitates were resolved by 8% SDS-PAGE under reducing conditions and immunoblotted with antiphosphotyrosine mAb (RC20H, BD Bioscience-PharMingen), anti-SHP-1 polyclonal rabbit antibody (Santa Cruz Biotechnology, CA), or streptavidin-HRP (Amersham Bioscience, Little Chalfont, UK).
Expression of soluble CD33 fusion proteins
For examining the correct expression of CD33m on human cells, soluble CD33M and CD33m molecules lacking the transmembrane and cytoplasmic regions were generated by transient transfection of the 293T human epithelial cell line. The cDNA encoding the CD33M or CD33m extracellular domains was generated by PCR using the forward ATC AGT AAG CTT CCT CAG ACA TGC CGC TGC and reverse AAC CAC TCC TGC TCT GGT CTC primers. The obtained PCR products (791 bp for CD33M or 410 bp for CD33m) were cloned in the topoisomerase cloning expression vector pcDNA3.1/V5/His (Invitrogen, Carlsbad, CA), placing the identification epitopes V5/His at the C terminus. Three CD33M or CD33m constructs (1 µg), m7 (from CD33m), M5 (from CD33M), and M8 (from CD33M), or control plasmid (pcDNA3.1) were transiently transfected into 293T cells by LipofectamineTM reagent (Invitrogen) following the manufacturer’s instructions. M8 was a spontaneous mutant from CD33M with Cys36-to-Arg substitution, which eliminates the intradomain disulfide bound in the V-Ig region according to Vullo and Frasconi [28
]. Supernatants from approximately 24 and 48 h of culture were harvested, and then, 293T-transfected cells were lysated. Supernatants and cell lysates were examined for the presence of CD33 fusion proteins on a Western blot by using HRP-conjugated anti-V5 mAb (Invitrogen). Supernatants and cell lysates were also immunoprecipitated with anti-CD33 mAb WM53-coated protein A-Sepharose CL-4B beads as described above.
Cytotoxicity assays
Redirected lysis assays were performed as described previously [24
]. Briefly, 51Cr-labeled P815 cells were preincubated for 15 min with 50 µl anti-CD16 (KD1, culture supernatant, NKL assays) or anti-CD3 (UCHT1, 1–0.1 µg/ml, T cell assays), together with mouse IgG1 (control isotype antibody, 5 µg/ml) or anti-CD33 (WM53, 5µg/ml), anti-ILT2 (HP-F1, culture supernatant), or anti-CD94 (HP-3B1, culture supernatant). After incubation, CD33+ NKL or activated T cells were added at different effector/target (E:T) ratios.
For the natural cytotoxicity assays, CD33+ NKL cells were cultured previously for at least 3 days in TCM supplemented with 10% heat-inactivated human normal serum (HNS) and rIL-2 (1000 U/ml). For these assays, anti-CD33 complete molecules and control IgG molecules were used as a result of the lack of commercial anti-CD33 F(ab')2 fragments. To prevent binding of the mAb to the Fc receptors (FcRs), NKL and K562 cells were preincubated in TCM supplemented with 10% HNS [29
]. Before coculture, NKL cells were coated with the anti-CD33 WM53 mAb (IgG1 isotype, 10 µg/106 cells) and cross-linked with a sheep F(ab')2 anti-mouse antibody (10 µg/106 cells) on ice. 51Cr-labeled K562 cells (5x103 cells per well) were cocultured with coated NKL cells at different E:T ratios in the presence of 10% HNS and rIL-2 (1000 U/ml). After 4 h incubation at 37°C, 100 µl supernatant was collected, and radioactivity was measured in a counter for the determination of 51Cr release and percentage of lysis. Coated NKL cells were used against 51Cr-labeled, noncoated NKL cells to test the effect of the mAb treatments on the cytotoxicity of NKL cells against themselves. In all experiments, the spontaneous release was less than 15% of maximal release.
NKL-K562 conjugation assays
NKL conjugation to K562 target cells was performed according to Rubio et al. [30
]. NKL cells were labeled green by incubation with 400 nM calcein acetoxymethylester (Molecular Probes, Eugene, OR) and coated with the antibodies as described above for cytotoxicity assays. K562 cells were labeled red with 235 µM hydroethidine (Molecular Probes). After coating, 5 x 104 NKL cells were seeded with different numbers of labeled K562 cells on a 96-well round-bottom plate to achieve different E:T ratios. Plated cells were centrifuged for 5 min at 500 rpm and incubated at room temperature for 20 min. Conjugation was stopped on ice until the percentage of conjugates was measured by flow cytometry. For each sample, a minimum of 8000 NKL cells was acquired. The frequencies of conjugation of effector cells () at different E:T ratios (R) were obtained by measuring the percentage of bound effector cells related to the total number of acquired effector cells. The binding isotherms were obtained by plotting versus 1/R (i.e., vs. the T:E ratio). The max and the index of conjugation of the area under binding isotherm (AUI) were calculated as parameters that allow expression of the overall binding efficiency for each treatment [30
].
Apoptosis detection assay
Apoptosis was detected on NKL cells by using the Annexin V-FITC apoptosis detection kit I (BD Bioscience-PharMingen). After coating NKL cells with WM53 or IgG1 (control isotype) mAb, cells were incubated at 37°C for 4 h, harvested, washed twice with cold PBS, and stained with Annexin-V and propidium iodide, according to the manufacturer’s instructions. Stained cells were analyzed by flow cytometry.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The phenotype of those CD3+CD33+CD14– cells was examined by flow cytometry and compared with their CD33– counterpart cells from the same cultures. As shown in Figure 1C , both populations were negative for other myeloid cell markers such as CD13 (not shown) and CD15 (Fig. 1C , panel B) or NK cell markers such as CD16 (Fig. 1C , panel C), CD56 (Fig. 1C , panel D), or CD57 (Fig. 1C , panel E). In contrast, CD33+ and CD33– T cell populations did express identical intensities of other cell-surface markers (T cell-specific or T cell activation markers), such as CD25, CD27, CD28, CD38, CD45RO, or CD95 (Fig. 1C , panels F–M). CD3+CD33+ and CD3+CD33– cells could be CD4+ or CD8+ T cells (Fig. 1C , panel F, and data not shown). Both populations were negative for the B cell marker CD19. Also, no phenotypic differences were found between CD33+ and CD33– T cells when using other cell-surface markers such as CD26, CD30, CD44, CD71, or CD103 (not shown). However, it was found that the CD33+ T cell population showed an appreciable lower expression of HLA-DR with regard to the CD33– T cells from the same culture (Fig. 1C , panel N), but this was not consistent in all cultures.
Some of the cell cultures stimulated under allogenic stimulus also contained a percentage of CD3–CD33+CD14– cells during the first 2 weeks of stimulation (Fig. 2 A ). This CD3–CD33+ non-T cell population could be ascribed as activated NK cells displaying the phenotype CD16+, CD25+, CD56+, and CD94+ (Fig. 2B , panel C). As seen on the T cell population, no phenotypic differences were found compared with their counterpart CD3–CD33– NK cells.
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As CD33 has been described as a myeloid-specific antigen [1
, 6
], we wanted to rule out that the observed expression of the CD33 protein could be a result of antibody cross-reaction. To exclude this possibility, different commercial anti-CD33 mAb were used to assess the expression of CD33 on activated T or NK cells, whole blood cells from different donors, and some CD33+ cell lines, such as U-937, LG-2, and NKL. The results of these assays (data not shown) revealed that all anti-CD33 mAb tested marked all myeloid cells [monocytes and U-937 (high labeling); granulocytes (dim labeling)] as well as the NKL cell line and activated NK cells. The mAb HIM3-4 did not stain the activated T cells from the different individuals tested, whereas all other anti-CD33 mAb tested displayed the same percentage of CD33+-activated T cells. In the same line of evidence, other CD33+ lymphoid cell lines, such as the B cell line LG-2 and the T cell line T-ALL 103/2, were not stained with HIM3-4 anti-CD33 mAb (data not shown). Double-staining of CD33+ U-937 cells with HIM3-4 and WM53 anti-CD33 mAb suggested the binding of these mAb to different epitopes. Although these data did not completely discard the possible cross-reaction of WM53 anti-CD33 mAb with another lymphocyte membrane receptor, they strongly suggest that the CD33 glycoprotein expressed on T and B lymphocytes may be structurally different from the CD33 glycoprotein expressed on myeloid or NK cells.
These results also indicate that human T and NK cells may express the CD33 antigen after appropriate activation signals and suggest that CD33 is not a myeloid-specific antigen, as it has been considered currently. The expression of CD33 may be maintained after T cell stimulation for a long period of time, probably playing some roles on the fate of the activated T cells.
The IP lymphoid CD33 antigen displayed a lower molecular weight
Next, IP assays using WM53 anti-CD33 mAb were performed to compare lymphoid and myeloid CD33 antigen. With this purpose, polyclonal-activated T cells and some TCC (all populations were 100% T cells but showed different percentages of CD33+ cells), the U-937 cell line (as a CD33-positive control), and LCL-GUS (as a CD33-negative control cell line) were biotin surface-labeled, immunoprecipitated with WM53 anti-CD33-coated protein A beads, and blotted with streptavidin-HRP. With this approach, under reducing conditions, we identified a single and wide band with an average mass of 75 kD on U-937 cells (Fig. 3
, lane 3), which corresponds to CD33 antigen [14
]. On polyclonal T cells and the TCC tested (Fig. 3
, lanes 1 and 2), the corresponding immunoprecipitate showed a lower molecular weight (67 kD). In contrast, no band could be detected on the negative control GUS (Fig. 3
, lane 4). The 67-kD band could also be immunoprecipitated from NKL and LG-2 lysates (see Fig. 9
, and data not shown).
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CD33+ cells coexpressed two isoforms of CD33 determined by RT-PCR
To correlate the CD33 expression pattern detected by flow cytometry and IP to the presence and size of the described CD33 mRNA, we performed RT-PCR analysis. We designed a pair of primers to amplify the complete ORF of CD33 cDNA, which was expected to have a size of 1385 bp. RT-PCR was performed on total RNA from several lymphoid or myeloid cell lines. Thus, we tested polyclonal human T cell lines obtained after two or more rounds of allogenic stimulation and displaying a different percentage of CD33+ cells, the CD33+ cell line U-937 as a positive control, and the B cell lines LG-2 and GUS as lymphoid CD33+ and CD33– cell lines, respectively. As shown in Figure 4 A
, all samples except the CD33– cell line GUS rendered the expected PCR product of 1385 bp, CD33M in the figure. It is surprising that a second PCR product was obtained from the same samples with a size of 976 bp (CD33m). These two PCR products were amplified from all cell lines displaying surface expression of CD33 antigen by flow cytometry, but no product was obtained from negative cells, such as the mouse cell line P815, the human B cell line Ramos, or the monocyte-depleted, fresh PBLs (Fig. 4B)
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1.4 and 1.6 kb have been described previously, although the differences between these two species have not been reported [6
, 7
]. To understand the pattern of CD33 expression observed by FACS, IP, and RT-PCR, human polyclonal-activated T cells as well as several human hematopoietic tumor cell lines were studied by Northern blot. We first used, as a probe for CD33, the PCR product of the CD33m isoform, which anneals with CD33M and CD33m putative mRNA. Using this probe, we found two different transcripts of a size lower than the 18-s fraction of the cellular RNA from U-937, NKL, and K562 cells, all of them expressing CD33 on their membranes (Fig. 6 A
). No annealing was obtained on the CD33– cell line GUS, while on polyclonal-activated T cells and on the LCL LG-2, both expressing membrane CD33, only the major transcript could be detected. As shown in the same figure, highly CD33-positive cells (U-937 and NKL) predominantly presented the larger transcript, contrary to K562 cells, where the smaller transcript was the majority form. Larger RNA species were also detected from all cell lines including CD33– cell GUS, probably representing, as a result of their size, other unprocessed transcripts of the CD33 gene.
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Altogether, these results indicate that the two transcripts detected by using the CD33m probe correspond to the two possible spliced variants of CD33, but only the larger one could correspond to the known isoform CD33M. The smallest one, which is scarcely represented in U-937 and NKL cells, may correspond to the new isoform CD33m described in this work, which is predominantly observed on K562 samples. In contrast, activated T cells and LG-2 cells may express only a trace amount of the CD33m transcripts not detected by Northern blot but detectable by RT-PCR. The presence of the largest transcripts in all cell lines tested (located at a 28-s level) suggests that a CD33 primary transcript could be expressed but not processed, indicating a regulatory step on the CD33 membrane expression at this point.
The lack of the V-Ig-like domain does not affect the correct expression of CD33m fusion protein
Although the CD33m form could be detected by the presence of its mRNA, we could not assess its presence as a membrane protein. This fact could be explained by the lack of an appropriate antibody to detect the CD33m form but also by the null expression at the cell membrane level. To investigate the last possibility, we tried to compare the expression of both proteins on the human cell line 293T, transiently transfected with plasmids containing the expression of the extracellular portion of CD33M or CD33m proteins bound to the V5/His-tag epitopes. Transfected cells were cultured for a maximum of 48 h, and supernatants from approximately 24 and 48 h were collected. Finally, transfected cells were lysed. Supernatants and cell lysates were tested in a Western blot for the presence of the V5 epitope. As shown in Figure 7 A
, the expression of CD33m (m7) and CD33M (M5) was found in the supernatants (22 and 44 h of culture) and cell lysates as protein bands of approximately 31 and 53 kDa, respectively. A mutant form of CD33M (M8), lacking the intradomain disulfide bond on the V-Ig-type domain, was found in cell lysates but was unable to be secreted to the extracellular medium, probably as a result of the incorrect folding of this mutant protein. No degradation signals were found in the cell lysates (Fig. 7A) . These results clearly indicated that the lack of the first domain on the CD33 protein, as found on the CD33m isoform, does not preclude its expression and secretion by the cells, and therefore, the membrane expression of the CD33m isoform is possible. Conversely, the correct expression of the m7 construct also may indicate that the interdomain disulfide bond that is found on all siglecs [5
] might not be necessary for the correct folding and expression of these proteins.
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Cross-linking of CD33 glycoprotein partially inhibits cytotoxic activity of NKL cells
A few CD33 functional studies have been performed but only on cell lines from myeloid origin [10
, 12
, 13
]. Conversely, it was described that CD33+ NK cells display lower cytolytic activity against target cells than their CD33– counterparts [20
]. To provide some evidence of the inhibitory capacity of CD33 molecules on lymphoid cells, we used our CD33+ NKL cell line or polyclonal-activated T cells on redirected cytotoxicity assays against the FcR+ P815 murine mastocytoma cell line.
P815 cells were killed efficiently by CD16-triggered NKL with mAb KD1. When anti-CD33 mAb was added to the assay, lysis was reduced (Fig. 8 A ). Other inhibitory mAb, such as HP-F1 (anti-ILT2) or HP-3B1 (anti-CD94) mAb, completely inhibited the anti-CD16-mediated lysis. Thus, under these conditions, CD33 appears to function as an inhibitory receptor on NK cells blocking the partially CD16-mediated pathway of stimulation.
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We also tested the effect of CD33 protein engagement on NKL cells on natural cytotoxicity assays against K562. Our results showed that WM53 anti-CD33-coated NKL cells consistently displayed lower cytotoxicity against K562 target cells than the control mouse IgG1-treated NKL cells or nontreated NKL cells. This effect was not a result of the death of NKL cells by apoptosis or by cytotoxicity induced on WM53-coated NKL against themselves (not shown), and the reduced cytotoxicity against K562 was not caused by the lack of conjugation between the effector and the target cells, as shown in Figure 8D
. Contrarily, the WM53-coated NKL cells displayed a higher percentage of NKL-K562 conjugates and a higher efficiency of conjugation (max 20.47 and AUI=53.4, respectively) than nontreated (max=17.7 and AUI=38.55)- or IgG1-treated NKL cells (max=12.74 and AUI=49.5).
Furthermore, we observed that CD33 protein was tyrosine-phosphorylated after pervanadate stimulation, and this stimulus resulted in recruitment of the tyrosine phosphatase SHP-1 in NKL and T lymphocytes (Fig. 9 ).
Altogether, these results indicated that CD33 triggering may deliver inhibitory signals acting as a regulatory receptor on ADCC and natural cytotoxicity exerted by NK cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Up to date, from the 11 siglecs described, only a few of them have been identified on some T cell subsets. Thus, only a small subset of memory CD8+ CD45RO+ T cells expressed siglec-7 [33
]. A weak expression of siglec-9 has also been described on small subsets of CD8+ and CD4+ human T lymphocytes from PBL [34
]. More recently, siglec-10 and some new spliced variants have been detected on human leukemic T cell lines by RT-PCR [35
]. The expression of most siglecs has not been tested on activated T lymphocytes. Here, we have tested the expression of CD33 on resting peripheral T/B/NK lymphocytes with negative results. Thus, by RT-PCR, we have found resting lymphocytes negative for CD33M and the new isoform CD33m. However, our data demonstrate that a wide subset of CD4+- and CD8+-activated polyclonal T cells and some activated NK cells expresses the siglec CD33. Although not all activated T cells express CD33, CD33+ and CD33– T cells similarly express the activation markers CD25, CD38, or CD45RO. Regarding NK cells, only a subset of them expresses the CD33 antigen in our cultures, but this also happens with other siglecs. Thus, siglec-9 was expressed by 50% of NK cells, depending on the donor [34
]. Siglec-7, the first sialic acid-binding receptor to be identified on NK cells, was expressed by a dominant population of resting NK cells, although it became down-regulated following NK cell activation [33
]. Altogether, our results suggest that siglec-3 could be the most important member of the siglec family expressed on activated T lymphocytes. Conversely, these findings suggest that some siglecs, such as CD33, may be implicated on the regulation of not only the innate but also the adaptive immunity.
Moreover, we found by IP that CD33 glycoprotein on lymphoid cells has a lower mass compared with their myeloid counterpart, probably as a result of lineage-dependent, post-translational modifications. Thus, the CD33 antigen expressed on monocytes and activated T lymphocytes could differ in its degree of glycosylation, as found for other membrane glycoproteins such as CD8, CD86, CD43, or CD44 [31 , 32 , 36 37 38 39 ]. Glycosylation has particular relevance in the immune system, where cell-surface proteins and lipids involved in immune recognition and regulation are modified by various glycan structures during cell development and activation [40 , 41 ]. Thus, as it has been described for the CD8ß chain, different sialylation, which is regulated by specific sialyltransferases, may affect the recognition capacity and the development of lymphocytes [38 , 39 , 42 ]. Several glycoforms of CD43 are known to regulate cellular interactions in the immune system. One such glycoform, the CD43, which bears core 2 O-glycans, is expressed on T lymphocytes and NK cells but only after their activation [31 , 32 ]. In the same way, the CD33 antigen, expressed by T lymphocytes, may differ from myeloid CD33 in their ligand-binding properties in cis and in trans, conferring lymphoid CD33 different functional characteristics [1 , 2 ].
The existence of the alternatively spliced form of CD33m represents a new element in the complexity of CD33 biology. Two CD33 transcripts of 1.4–1.5 and 1.6–1.8 kb were identified in a panel of human myeloid leukemia cell lines by Simmons and Seed [7 ] and Peiper et al [6 ] with a different predominance on different cell lines (U-937, HL-60, or K562). The smallest transcript (1.4–1.5 kb) was suggested to be the approximate correspondent from the CD33 cDNA because of its similar size [7 ]. By using two different probes, which differentiate between the two CD33M and CD33m transcripts, we demonstrate here that the larger transcript corresponds to the known CD33M mRNA, and the smallest one is the correspondent for the CD33m transcript. Although CD33m is detectable by RT-PCR, the CD33m mRNA may be present in low amounts in T and B lymphoid cell lines used in our work. The clear presence of CD33m transcripts in U-937, NKL, and K562 suggests a functional role of CD33m only on myeloid and NK cells.
CD33 may be expressed as a homodimer on the cell membrane [43 ]. The novel isoform CD33m conserved the cytoplasmic domain but lacked the external V-Ig domain where the ligand-binding site is located. Thus, heterodimers formed by CD33M–CD33m could exist with a lower capacity of ligand binding but with the same level of regulatory function as compared with CD33M–CD33M homodimers. At the present time, we have no evidence of the CD33m membrane expression, probably as a result of the lack of specific antibody against CD33m isoform. However, mAb are now being developed in our laboratory with the ability to recognize and differentiate both isoforms.
CD33 leukocyte antigen has been identified as a potential inhibitory receptor on myeloid cells with differential ITIM function in recruiting the phosphatases SHP-1 and SHP-2 [9 10 11 12 13 ]. Our experiments on NKL cells reveal that engagement of CD33 partially reduces the lytic capacity of those cells in the rADCC assays against P815 and against their natural target K562. CD33 protein was tyrosine-phosphorylated, and SHP-1 tyrosine-phosphatase was recruited after pervanadate stimulation in NKL and T cells. We propose that CD33 is acting as an inhibitory receptor on lymphoid cells. Thus, the detection of the sialic determinants recognized by CD33 on target cells could modulate the natural cytotoxic activity of the NK cell, as recently seen for siglec-7 [44 ].
Sialic acids are expressed abundantly in mammals and are commonly found at the cell surface and in the extracellular environment, attached to a wide variety of proteins and lipids. Their role in cell–cell repulsion and masking of subterminal sugars is well known [45 46 47 ]. They are present on most of the self-antigens but absent from many potential pathogens [45 46 47 ]. Thus, the hypothesis that sialic acids have evolved to function as molecular determinants of "self" is emerging [5 ]. The carbohydrate structure attached to glycoproteins may be altered as a result of several events, such as cell differentiation or activation [31 , 32 , 48 ]. Sialylation may also decrease during infection, as detected on mucins from rat small intestine when infected by the intestinal parasite Nippostrongylus brasiliensis, being modulated by the expression of specific enzymes during the infection process [49 ]. These events or the exposure of the cell surface to sialydases produced by some invaders (e.g., influenza virus or some bacteria [50]) would decrease the sialic acid contents, which could engage the siglecs. Thus, the surface expression and the negative signals triggered by CD33 on T lymphocytes and NK cells could contribute to the setting of the appropriate activation thresholds by interacting with their sialylated ligands on APC or target cells. This could help prevent undesirable self-reactivity and tissue damage and at the same time, allow effective killing of nonsialylated targets. Conversely, CD33 may recognize as ligand the sialyl-Tn, an antigen, which is expressed on different tumors with poor prognosis [51 ]. Thus, CD33, on T cells, could contribute to the surveillance of these tumors, eventually leading to modulation of the activation stage of the effector cells.
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ACKNOWLEDGEMENTS |
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Received February 17, 2005; revised July 30, 2005; accepted August 24, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pigment Cell Res 13,120-126[Medline]
) or IgG1 control isotype antibody (
) and cross-linked with F(ab')2 sheep anti-mouse antibody on ice in the presence of 10% HNS. Noncoated (control cells, 
) and coated NKL cells were analyzed in a cytotoxicity assay against K562 cells at different E:T ratios in the 51Cr cytotoxicity assay. The figure shows one experiment out of four with similar results. Each point represents the average of triplicate values. The range of the triplicates was within 5% of their mean. (D) The binding of the NKL-K562 E:T system was studied as described in Materials and Methods. After staining, NKL cells were treated as above and cultured with different amounts of K562 cells as described for cytotoxicity assays. Percentages of NKL-K562 conjugates related to the total NKL cell tested (