ARTICLE |
Correspondence to: Georges Delsol, Unité de Physiopathologie Cellulaire et Moléculaire, CNRS-UPR 2163, Institut Claude de Preval, IFR 30, Chu de Purpan, 31059 Toulouse, Cedex 03, France. E-mail: rubin@immgen.cnrs.fr
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Summary |
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We generated a new monoclonal antibody (MAb), F7.2.38, by immunizing mice with CD3/CD3
complexes purified from human T-cells by OKT3 MAbSepharose affinity chromatography. Immunoprecipitation experiments and Western blotting analysis showed that MAb F7.2.38 recognized the CD3
chain in CD3
cDNA-transfected FOX B-cells and in various T-cell lines. Using flow cytometry on permeabilized or intact cells, the epitope was found to be located in the cytoplasmic tail of the CD3
chain. Immunohistochemical staining on paraffin-embedded sections showed that the reactivity of MAb F7.2.38 was comparable to that of the commercially available anti-CD3
polyclonal antibody. Of the 52 well-characterized T-cell lymphomas, 41 were positive for F7.2.38 (79%), whereas all 37 B-cell lymphomas and 69 non-lymphoid tumors were unreactive. This new anti-CD3
antibody would be particularly useful for phenotyping T-cell lymphomas on routinely processed paraffin-embedded tissue sections. (J Histochem Cytochem 48:16091616, 2000)
Key Words:
monoclonal antibody, CD3 chain, T-cell lymphomas, T-cell receptor assembly, immunohistochemistry, paraffin sections
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Introduction |
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THE T-CELL RECEPTOR is composed of two clonotypic chains, TCR and TCRß, non-covalently associated with the invariant chains called CD3
, CD3
, CD3
, and
(
or TCRß variable and constant regions, or external and cytoplasmic domains of CD3
chain, have been produced by immunizing mice, rats, or hamsters with human T-cell clones or lymphomas. The anti-CD3
MAbs are directed against two different types of epitopes: (a) unique "conformational epitopes" created in CD3
or CD3
heterodimers, and (b) epitopes on isolated CD3
chains (
or CD3
chains have never been obtained. This could be due either to amino acid sequence similarity between CD3 molecules of donor and recipient species or to lack of accessibility of the CD3 molecules to the immune system. However, there appear to be ample differences in amino acid sequences between CD3
and CD3
molecules from human and mouse (see
These considerations have led us to develop methods for the production of MAbs against all components of known human TCR/CD3 complexes (pre-TCR, TCR, or TCR
ß). Such MAbs will be very useful for studies on TCR/CD3 structure and in the characterization of TCR complexes on T-lymphocytes in various diseases such as immunodeficiency, allergy, autoimmunity, and cancer. Our goal has been to produce MAbs useful in many different analytical systems, i.e., cytofluometry on living or permeabilized cells, immunohistochemistry on tissue sections prepared by different methods, immunoprecipitation in different detergents, and Western blotting analyses. Because immunization with living T-cells has been unsuccessful, we attempted to isolate CD3 complexes from NP40-T cell lysates on anti-CD3
MAb affinity columns. Mice were injected with the eluted material, which contained mainly CD3
,
, and
molecules, plus an intracellular chaperone, CD3
, implicated in CD3 complex assembly (
Here we present the characterization of one of the MAbs obtained using this strategy. The MAb, F7.2.38, directed against a cytoplasmic epitope in the CD3 chain, recognizes an epitope present in both frozen and formalin-fixed material. The reactivity of this antibody was compared with that obtained using the commercially available rabbit anti-CD3
antibody (Dako; Trappes, France).
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Materials and Methods |
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Production of MAb F7.2.38
CD3 complexes were obtained by affinity chromatography of 2% NP-40 detergent (Fluka Chemie; Buchs, Switzerland)-solubilized proteins from Jurkat T-cells (250500 x 106 cells/immunization/mouse) on a 5-ml Sepharose bead column (Amersham Pharmacia Biotech; Orsay, France) with covalently coupled MAb OKT3. Elution was performed with 1 M NH4SCN or 0.2 M glycine buffer, pH 2.8. F1 hybrid female mice (C57B6 x Balb/c) were immunized three times, once SC and twice IP, with eluted material in 250 µl of DPBS (NaCl 0.14 M, Na2HPO4 8 mM, KH2PO4 1.5 mM, KCl 2.7 mM, pH 7.2) with 250 µl of complete Freund's adjuvant (Statens Seruminstitut; Copenhagen, Denmark). Four months later the mice were immunized regularly (at least five times at 2-week intervals) with IP injections of equal doses of the CD3 complexes, still adsorbed onto the beads, without adding Freund's adjuvant. All mice produced antibodies precipitating CD3, CD3
, CD3
, and CD3
proteins as well as molecules with higher (>30,000 Daltons) or lower (<20,000 Daltons) molecular weights (not shown). Five days after the last IP immunization, fusion of the spleen cells with the non-Ig-producing myeloma cell line X63 Ag8 653 was performed using standard techniques (
, CD3
, CD3
, and CD3
chains.
Cell lines and MAbs
Human leukemia ß (Jurkat subclone E6.1, HBP-ALL, CEM, and MOLT4) and
(LYON and PEER) T-cells, human B-lymphoma Raji cells, and murine B-lymphoma FOX cells were grown in RPMI 1640 with L-Glutamate supplemented with 1 mM sodium pyruvate, penicillin/neomycin/streptomycin mixture (Life Technology; Eragny, France), and 8% of fetal calf serum (FCS) (Roche; Meylan, France).
Mouse SP-34, OKT3, and F101.01 MAbs specific for single or CD3-associated CD3 components, respectively, were obtained from Dr. C. Terhorst (Boston, MA), ATCC (Rockville, MD), and Dr. T. Plesner (Copenhagen, Denmark). CD20/L26 anti-B cell MAb and the rabbit polyclonal anti-CD3
(A0452) were purchased from Dako.
Flow Cytometric Analyses
Cells were permeabilized using 0.1% saponin (Sigma; Saint Quentin Fallavier, France). A total of 106 cells were washed in DPBS and labeled for 30 min at 4C with F7.2.38, SP-34, and OKT3 MAbs supplemented with 0.1% saponin. After two washes in DPBS containing 0.1% saponin, cells were incubated for 30 min at 4C with a fluorescein isothiocyanate-conjugated rabbit Fab2' anti-mouse (F0313; Dako) supplemented with 0.1% saponin. Cells were washed in DPBS with 0.1% saponin and analyzed with the Coulter Electronics Cytometer (Coulter EPICS XL; Hialeah, FL).
For surface staining analysis, exactly the same procedure was followed, but without adding saponin, and using a phycoerythrin-conjugated goat anti-mouse (IM0551; Immunotech, Marseille, France) as secondary antibody.
Transfections
Murine B-lymphoma FOX cells were transfected by electroporation. Approximately 5 x 106 cells were washed twice in culture medium without FCS, resuspended in the DNA transfection mixture [5 µg of pSRa-CD3del12 described by B. Alarcon (
MAb SP-34. In this report, all results shown were obtained with clone T116.4.3.
Western Blotting Experiments
A total of 5 x 106 FOX, T116.4.3, or Jurkat E6.1 cells were washed twice in DPBS and then lysed in 500 µl of Tris 0.5 M, pH 6.8, 2% SDS, 10% glycerol, bromophenol blue, with or without 5% ß-mercaptoethanol. After three 5-sec sonications, lysates were boiled for 10 min at 100C and centrifuged for 10 min at 14000 x g, 4C. Thirty µl of supernatant was separated by SDS-PAGE (12%) according to the
Immunostaining of Various Human Normal and Neoplastic Tissue Sections
F7.2.38 supernatant diluted 1:10 was used to study its reactivity on a large panel of a variety of normal and pathological human tissue samples from the tissue bank of the department of Pathology at Purpan Hospital in Toulouse, using the multi-tissue block method (
Different human tumors (n = 179) were investigated. These included: (a) 110 cases of lymphoid neoplasms [including 89 non-Hodgkin's lymphomas classified according to the REAL and the WHO classifications (
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Paraffin-embedded sections were immunostained by the streptavidinbiotinperoxidase complex (ABC) method using Dako StrepABComplex/HRP Duet (mouse/rabbit) kit (K0492; Dako). The antigen retrieval method used heat pretreatment of sections in a 750-W microwave oven (10 min of boiling twice in citrate buffer, pH 6) (
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Results |
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Characterization of the Molecule Targeted by MAb F7.2.38
Similar Patterns of Proteins Precipitated by Known Anti-CD3 and F7.2.38 MAbs.
MAb F7.2.38 was selected because it could (a) recognize the human leukemia T-cell line Jurkat by intracellular staining analyzed by flow cytometry and (b) immunoprecipitate proteins with molecular weights similar to those of the CD3
, CD3
, CD3
, and CD3
chains from 2% NP-40 lysates of Jurkat cells. All TCR/CD3 proteins except CD3
are usually co-precipitated with an anti-CD3
MAb from a 1% digitonin T-cell lysate. However, if 2% NP-40 is used, only CD3
,
,
, and
chains are co-precipitated. Therefore, comparable immunoprecipitations with MAb F7.2.38 of either digitonin or NP-40 lysates may indicate whether the MAb is specific for CD3
or CD3
molecules. The results in Fig 1 demonstrate that MAb F7.2.38, like MAb OKT3, co-precipitated three bands corresponding to CD3
and CD3
from NP-40 lysates and no CD3
molecules from digitonin lysates. Consequently, MAb F7.2.38 appeared to react against the CD3
chains rather than CD3
. One difference between the immunoprecipitations with MAbs OKT3 and F7.2.38 was the presence of an additional band of 40 kD in the F7.2.38 immunoprecipitates (arrow). This 40,000-Dalton band was also present in immunoprecipitates with the SP-34 MAb specific for single CD3
chains (data not shown). The same results were obtained when we used MAb F7.2.38 with lysates from other human T-cell lines, such as the
ß HBP-ALL, CEM, and MOLT4 T-cell lines or the
LYON and PEER T-cell lines (data not shown). MAb F7.2.38 did not immunoprecipitate any TCR/CD3-like proteins from Raji B-cell lysates (Fig 1). These data indicate that MAb F7.2.38 reacts similarly to some other anti-CD3
MAbs in both detergents used.
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MAb F7.2.38 Specifically Recognizes the Human CD3 Chain.
These results led us to postulate that MAb F7.2.38 could be directed against the CD3
chains or against CD3
or CD3
dimers. Therefore, we transfected the murine B-cell lymphoma FOX with a cDNA encoding a truncated CD3
chain (
epitope (see Introduction). To confirm these results, a Western blotting experiment was carried out using lysates of the CD3
-expressing FOX cells and blotting the membranes with MAb F7.2.38 or SP-34. Fig 3 shows that a
40-kD band was revealed by MAb F7.2.38 in T116.4.3 cell lysates but not in FOX cell lysates. The proteins in this band were reduced (in the presence of ß-mercaptoethanol) to a
20-kD band, indicating that the 40-kD band represented a disulfide-linked homodimer of CD3
chains. In E6.1 cell lysates, MAb F7.2.38 revealed a 20-kD band and a 40-kD band. As before, the 40-kD molecules (but not the 20-kD molecules) were reduced to 20 kD. This 40-kD band appears similar to a corresponding molecule in Fig 1 (arrow). The same pattern was observed when the membranes were blotted with MAb SP-34 (data not shown). Similar results were obtained when immunoprecipitation experiments were performed with lysates of [35S]-methionine/cysteine-labeled cells and MAb SP-34 or F7.2.38. However, the results obtained with MAb F7.2.38 proved more constant than those obtained with MAb SP-34 in Western blotting analysis. In contrast, MAbs F7.2.38 and SP-34 demonstrated equivalent activities against CD3
molecules in [35S]-methionine/cysteine-labeled cell lysates (data not shown).
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MAb F7.2.38 Does Not Recognize the CD3 Chain on the Surface of Cells.
The cDNA used for FOX cell transfection experiments encodes the CD3
chain with a deleted endoplasmic reticulum retention signal sequence, which in principle should allow the molecule to be expressed at the cell surface (
chain and the E6.1 CD3
chain (Fig 3). However, cell surface staining was detected on T116.4.3 and E6.1 cells with MAb SP-34 but not with MAb F7.2.38 (Fig 2B). Therefore, unlike MAb SP-34, this MAb is most likely directed against the cytoplasmic region of the CD3
chain.
Reactivity of MAb F7.2.38 on Paraffin-embedded Tissue Sections
Normal Human Tissues.
In normal lymphoid tissue, MAb F7.2.38 showed strong staining of lymphoid cells in the medulla and cortex of normal thymus (Fig 4A). As expected, only scattered B-lymphocytes in the cortex and medulla were labeled with MAb CD20/L26 (Fig 4B). In human lymph node and tonsil, interfollicular areas containing the T-lymphocytes were strongly stained, well delimiting B-cell follicles in which some small reactive T-cells were labeled (Fig 4C). This reactivity was complementary with immunostaining of B-cell zones using MAb CD20/L26 in lymph node tissue (Fig 4D). With the exception of scattered T-cells, MAb F7.2.38 was unreactive with all other normal human tissues studied (data not shown).
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Immunostaining on frozen and paraffin-embedded tissue sections was comparable, and therefore only paraffin sections were used in further experiments. Almost all results obtained with MAb F7.2.38 were comparable to the reference antibody, the rabbit polyclonal anti-CD3 antibody.
Neoplastic Human Tissues
Lymphoid Tumors.
The reactivity of MAb F7.2.38 with lymphoid tumors is shown in Table 1. All B-cell lymphomas were negative for F7.2.38 (0+/37), and only small reactive T-cells were strongly positive with this antibody. On the contrary, the majority of T-cell lymphomas [41+/52 (79%)] were positive for MAb F7.2.38 (Fig 4E4H). As expected, anaplastic large-cell lymphomas (ALCLs) showed a lower rate of immunostaining with this antibody [6+/14 (43%)] in comparison with the other types of T-cell lymphomas [35+/38 (92%)] (Table 1) (Fig 4E4G). In this group of T-cell lymphomas, only 6/14 cases studied showed a positive staining of a variable percentage of neoplastic cells for F7.2.38. The labeling intensity was also variable from case to case and overall was lower than that obtained in the other types of T-cell lymphomas. It is noteworthy that in 2/6 positive ALCLs, only rare neoplastic cells showed weak cytoplasmic staining with MAb F7.2.38. Of note was the reactivity of MAb F7.2.38 with nasal type extranodal NK/T-cell lymphoma. In this last category, 3/5 cases studied were positive for MAb F7.2.38 (Table 1) and virtually all neoplastic cells were labeled (data not shown). Lastly, among the 21 cases of Hodgkin's disease of different categories, only two cases (9.5%) showed focal cytoplasmic staining of a variable number of Hodgkin and ReedSternberg cells. In the other cases, neoplastic cells were clearly negative and were often surrounded by strongly labeled small T-lymphocytes.
Non-lymphoid Tumors.
The 69 non-lymphoid tumors investigated were always negative for MAb F7.2.38, which stained only small reactive T-cells (data not shown). The same results were obtained with the polyclonal anti-CD3 antibody.
The F7.2.38 antibody worked well on paraffin sections stained either manually, or with either of two automated immunostainers (TechMate 500 and Ventana 320/ES).
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Discussion |
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In this article we have described a novel MAb, F7.2.38, which recognizes the human CD3 chain, associated or not with the TCR/CD3 complex. This MAb can be used for all biochemical and immunohistochemical methods in T-cell receptor research and in routine clinical laboratory tests. Our results show that MAb F7.2.38 recognizes both monomeric and dimeric forms of the CD3
chain (see Fig 3). The latter form is dominant in the CD3
cDNA-transfected non-T-cells compared to normal T-lymphocytes, in which all components of the TCR/CD3 complex are present. These results are in agreement with those described by others (
The main result from this study is that MAb F7.2.38 is directed against a fixative-resistant epitope of the CD3 polypeptide. Therefore, it can be used to identify normal and neoplastic T-cells on paraffin-embedded tissue sections. The reactivity of the polyclonal anti-CD3
antibody on routinely processed biopsy specimen made it a valuable reagent for identification of T-cell lymphoid malignancies. The reactivity of MAb F7.2.38 was very similar to that of the routinely used polyclonal anti-CD3
antiserum. It is of interest that almost all T-cell neoplasms other than anaplastic large-cell lymphoma express CD3 antigen on the surface and/or in the cytoplasm, whereas B-cell lymphomas are invariably negative. As previously reported (
antibody in approximately one third of cases. In addition, in this group of T-cell lymphomas the staining intensity and the percentage of positive cells are always lower than those observed in the other T-cell lymphoma subtypes. It is also of interest to note the cytoplasmic staining of three out of the five nasal type extranodal NK/T-cell lymphoma cases. As previously reported (
in paraffin sections despite the lack of CD3 detection on the cell membrane. This latter finding is likely consistent with the cytoplasmic expression of CD3
chain by the majority of NK-cells, from which these lymphomas originate (
reactivity of ReedSternberg cells in some cases appears as a globular and not as a diffuse cytoplasmic staining, as previously reported (
Until now, the polyclonal anti-CD3 has been the most commonly used antibody in immunohistochemistry on paraffin sections, giving specific and intense staining of reactive and neoplastic T-cells (
chain is suitable for use on routinely fixed and paraffin-embedded tissues. This is probably due to the fact that the vast majority of these MAbs (see
chain, which could be easily destroyed by fixation of tissues. Interestingly, both the polyclonal anti-CD3
and the F7.2.38 mAb appear to recognize the cytoplasmic region of the CD3
. This unique reactivity provides a valuable tool for functional and immunohistochemical studies. In the present study, we noted that MAb F7.2.38 worked as well as the polyclonal anti-CD3
in immunohistochemical methods, either manually or using automated immunostainers.
In summary, we have produced a novel MAb directed against the human CD3 protein, which would be very useful in studies on TCR/CD3 complex assembly as well as in the immunohistochemical diagnosis of T-cell lymphomas.
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Acknowledgments |
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Supported by institutional grants from the CNRS, l'Université Paul Sabatier (UPS) Toulouse, and l'Association pour la Recherche contre le Cancer (ARC).
We thank Pr Bill Clark (UCLA) for valuable comments on the manuscript, Dr Balbino Alarcon for the kind gift of the vector pSRa-CD3del12, and Jeanine Boyes, Françoise Dupic, and Dr Andres Alcover for their excellent technical advice. We acknowledge the initial help of Cécile Gouaillard in this project.
Received for publication June 16, 2000; accepted August 10, 2000.
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