BMA031, a Monoclonal Antibody Suited to Identify the T-Cell Receptor a /CD3 Complex on Viable Human T Lymphocytes in Normal and Disease States

Jannie Borst, Jacques J. M. van Dongen, Evert de Vries, W. Marieke Comans-Bitter, Maarten J. D. van Tol, Jaak M. Vossen, and Roland Kurrle

A B S T R A C T : Two types o f T lymphocytes can be discriminated on the basis of expression of either the classical T-cell receptor (TCR) ~[3 or the more recently identified TCR 78. Whereas TCR ~ + lymphocytes are known to respond to recognition of antigen in the context of major histocompatibil- ity complex molecules by proliferation, ly'mphokine secretion, and~or cytotoxicity, the potential ligand specificities and functions of TCR 78 + cells have not been completely unraveled. Antibodies specific for either receptor type are important tools to elucidate the role TCR 78 + cells play in the immune system. They can be used to quantify TCR 78+ cells and TCR ~[3 ÷ cells in normal and disease states, to isolate both T-cell subsets, and to perform in vitro functionaf assays. Only few antibodies reactive with common determinants on either TCR ~[3 or TCR 78 are available. Generally, the monoclonal antibody (mAb) WT31 is used for definition of viable human TCR ~ + cells. However, W T 3 I has recently been shown to cross-react with TCR 7& We describe an mAb, BMA03I , that combines the unique features of reactivity with intact viable cells and true specificity for a common determinant on the TCR ~[3/CD3 complex. Its performance in immunofluorescence staining and immunochemistry has been compared with that of WT31 and anti-TCR 78 mAbs, using TCR ~13 and TCR 7~ expressing cells isolated from blood and bone marrow of healthy individuals and immunodeficient patients.

A B B R E V I A T I O N S BM BSA CHAPS

FITC IF

bone marrow IPB bovine serum albumin MHC 3-(3-cholamidopropyl)

dimethylammonio- 1- mAb propanesulfonate MNC

fluorescein isothiocyanate NP-40 immunofluorescence PB

immunoprecipitation buffer major histocompatibility

complex monoclonal antibody mononuclear cells Nonidet-P40 peripheral blood

From the Division of Immunology, The Netherlands Cancer Institute, Amsterdam (J.B.; E.d.V.), Depart- ment of lmmunology, Erasmus University, Rotterdam (J.J.M. v.D., W.M.C.-B.), Department of Pediatrics, Sophia Children's Hospital, Rotterdam (W.M.C.-B.), Department of Pediatrics, University Hospital, Leiden (M.J.D.v.T., J.M.V.), The Netherlands, and Research Department for Biochemistry and Immunology, Behringwerke AG, Marburg, Federal Republic of Germany (R.K.).

Address reprint requests to Dr. Roland Kurrle, Research Department for Biochemistry and Immunology, Behringwerke AG, P.O. Box 1140, 3550 Marburg/Lahn, FRG.

Received September 18, 1989; accepted April 20 1990.

Human Immunology 29, 175-188 (1990) 175 © American Society for Histocompatibility and Immunogenetics. 1990 0198-8859/90/$3.50

176 J. Borst et al.

PBS phosphate-buffered saline TCR T-cell receptor PE phycoerythrin TR1TC tetramethylrhodamine SDS-PAGE sodium dodecylsulphate- isothiocyanate

polyacrylamide gel electrophoresis

I N T R O D U C T I O N

Two types ofT-cell receptors (TCR) are now know. TCR a/3 is expressed on the majority ofnormal human T lymphocytes (>80%) and recognizes foreign antigens in the context of molecules of the major histocompatibility complex (MHC) [ 1]. TCR y~ is expressed on a minority of human T cells in peripheral lymphoid organs and blood, as well as in most epithelial tissues (<20%, with great variation between individuals) [2-7].

To understand what contribution TCR y8 ÷ cells make to the immune system, it is important to analyze human disease states where T lymphocytes are thought to play a role, especially in cases ofautoimmunity, tissue transplantation, infectious diseases, and cancer. Quantification of the relative contribution ofTCR a/~ + and TCR y8 + cells to T-cell infiltrates in disease lesions, followed by isolation of these cell populations and in vitro functional analyses may help elucidate the function of TCR y8 + cells. Specific antibodies are indispensable tools in such studies.

TCR a/3 and TCR y8 are both composed of two chains, each with a variable and a constant domain [8,9]. TCR a/3 was first defined at the protein level by monoclonal antibodies (mAbs), reactive with clone-specific epitopes on the vari- able domain [1]. Since then, many clonotypic, and also V3-specific mAbs have been described [ 10-17]. However, to our knowledge only two mAbs are available that seem directed at common determinants on TCR a/~ and react with intact viable cells: WT31 [18,19] and BMA031 [20-23]. Other reagents, such as the mAbs aF1, aF2 [24],/3F1 [25], the rabbit antisera H36 (anti-TCR a chain), H38 (anti-TCR/3 chain) [26], anti-TCR Ca, and anti-TCR C/32 [27] were raised against purified denatured protein or synthetic peptides and do not react with the native TCR. Human TCR ~/8+ cells were first identified as cells reactive with anti-CD3, but not with WT31 mAb [28,29]. Since then two mAbs have been generated reactive with common determinants on TCR y8 [3,30], which can be used success- fully to quantify and isolate viable TCR y8 + cells.

Until now, most researchers have used the WT31 mAb for quantification and isolation of TCR a/3 + cells. However, WT31 mAb may cross-react with TCR y8 [31], which can lead to misinterpretation of results, particularly in disease states as will be demonstrated here. This article describes BMA031 mAb as the only reagent available thus far that is truly specific for a common determinant on the native TCR afl/CD3 complex.

METHODS

Cells. Mononuclear cells (MNC) were isolated from peripheral blood (PB) and bone marrow (BM) by Ficoll-Isopaque differential density Centrifugation. Cell samples were obtained from healthy individuals (60 PB samples) and from immu- nodeficient patients (84 PB samples and 6 BM samples), including patient P.B., suffering from Wiskott-Aldrich syndrome. Cloned lymphocytes were generated from PB of healthy individuals in case of AK 4 (donor X3), 1012 (donor YS), JS-93, - 132, -318, -332 (donor JS), and JoBo-22 (donor JoBo). Clones AK 4 and JoBo-22 were derived from PB-MNC directly by limiting dilution, clone 1012 from CD4-8 sorted PB-MNC, clones JS-318 and -332 from TCR y~+ sorted PB- MNC [32], and JS-93 and -132 from a mixed lymphocyte culture enriched for

BMA031: mAb Specific for Human TCR ~/3/CD3 177

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. . . . . . .

JS-132

loo,

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JS-332

JS-318

PEER

• Fluorescence Intensity

FIGURE 1 Comparison performance in IF on BMA031 and WT31 mAb on activated T cells. T-cell clones were incubated with anti-CD3, anti-TCR ~/~-1, WT31, and BMA031 mAbs, followed by FITC-conjugated goat anti-mouse Ig. Clone JS-132 expresses TCR ~/3, clone JS-332 disulphide-linked TCR ~8, clone JS-318 and the cell line PEER non-disul- phide-linked TCR ~/~.

cells expressing a V~5-encoded determinant [ 13,14]. All clones were cultured in Yssel's medium [33], supplemented with 2% pooled human serum in round- bottom microwell plates (Greiner Labor Technik, N~irtingen, FRG) in the pres- ence of phytohemagglutinin, irradiated allogeneic PB-MNC, and Epstein-Barr virus transformed human B cells of the line JY (feeder cells). The JY cell line and the human T leukemic cell line PEER [34] were cultured in Iscove's medium with 5% fetal calf serum under standard conditions.

Antibodies. Anti-TCR o~ mAb BMA031 (IgG2b) was obtained by immunization of Balb/c mice with human-erythrocyte-rosette-positive lymphocytes [20] and is available from Dr. R. Kurrle, Behringwerke, Marburg/Lahn, FRG. Anti-TCR ~ mAb WT31 (IgG1) has been raised against human thymocytes [18] and was a kind gift from Dr. W. Tax (University of Nijmegen, The Netherlands). Anti-TCR ~/8-1 mAb (hybridoma 11F2; IgG1) was raised against purified TCR ~/6/CD3 complex [3]. Anti-TCR 61 mAb [30] (IgG1) was from T Cell Sciences (Cam- bridge, MA). Of anti-CD3 mAbs, SPV-T3b [35] (IgG2a) was used for single and double immunofluorescence (IF) stainings (Figs. 1 and 2), VIT-3 (IgM; a gift from Dr. W. Knapp, University of Vienna, Austria) for double IF stainings (Fig. 3), and 2G3 (IgG2a; donated by Dr. Zhang Jingwu, Dr. Willems-Instituut, Diepenbeek, Belgium) for immunoprecipitation (Fig. 4).

Immunofluorescence. Indirect single IF stainings were performed according to stan- dard procedures at 4°C in phosphate buffered saline (PBS), contai,i~g 0.5% w/v bovine serum albumin (BSA) and 0.02% w/v sodium azide. In single IF

178 J. Borst et al.

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PB-MNC were isolated and incubated with first mAb, followed by incubations with PE- conjugated anti-mouse Ig, normal mouse Ig, and FITC-conjugated second mAb. The numbers in each quadrant indicate the percentages of stained cells. (A) FITC-labeled anti- TCR ~/~-1 versus PE-labeled anti-CD3 mAb. (B) FITC-labeled BMA031 versus PE-labe|ed anti-CD3 mAb. (C) F1TC-labeled anti-TCR 78-1 versus PE°labeled BMA031 mAb. (D) FITC-labeled WT31 versus PE-labeled anti-CD3 mAb.

stainings (Fig. 1), fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse F(ab')2Ig (TAGO, Burlingame, CA) was used as second step reagent. Double IF stainings shown in Table 1 were performed as follows: for double labeling with anti-TCR (WT31, BMA031, or anti-TCR y8-1) and anti-CD3 mAb (anti-Leu-4, Becton Dickinson), cells were incubated with biotin-conjugated anti-TCR mAb, followed by FITC-conjugated streptavidin and phycoerythrin (PE)-conjugated anti-CD3 mAb; for double labeling with WT31 or BMA031 on the one hand and anti-TCR ~/8-1 on the other, cells were incubated with FITC-conjugated WT31 or BMA031 mAb, followed by biotin-conjugated ant!-TCR ~8-1 and PE-conjugated streptavidin. For double IF stainings of Fig. 2, cells were incubated with first mAb, followed by PE-conjugated anti-mouse Ig (Becton Dickinson), normal mouse serum, and FITC-conjugated anti-TCR y8-1, BMA031, or WT31 mAb. A FACScan (Becton Dickinson) was used for analysis. For double IF stainings of Fig. 3, WT31 and BMA031 were used to detect TCR ~3, anti-TCR T~-I to detect TCR 7~, and VIT-3 to detect CD3. Cells were first incubated with one of the

BMA031: mAb Specific for Human TCR ~ /CD3 179

anti-TCR mAbs (IgG) as well as with VIT-3 (IgM), followed by FITC-conjugated goat anti-mouse IgG serum and tetramethylrhodamine isothiocyanate (TRITC)- conjugated goat anti-mouse IgM serum (Nordic Immunological Laboratories, Tilburg, The Netherlands) to detect TCR expression and CD3 expression, respec- tively. After labeling, cytocentrifuge preparations were made (Cytofuge; Nordic Immunological Laboratories) and analyzed with a fluorescence microscope (Zeiss, Oberkochen, FRG). In each preparation, at least 500 CD3 + cells were evaluated for TCR ~/~ and TCR ~/8 expression.

Immunoprecipitation. About 20 × 106 viable cells were resuspended in PBS and labeled with i mCi Na125I (Amersham Co., Amersham, UK) using lactoperoxidase as a catalyst. After labeling, cells were washed several times in serum-free medium and lysed in immunoprecipitation buffer (IPB), consisting of 0.01 M triethanolam- ine-HCl, pH 7.8, 0.15 M NaCl, 5 mM EDTA, 1 mM PMSF, 0.02 mg/ml ovomu- coid trypsin inhibitor, 1 mM TLCK, and 0.02 mg/ml leupeptin, supplemented with detergent as indicated in the Results section. Nuclear debris was removed by centrifugation for 15 min at 13,000 × g. Lysates were centrifuged for 30 min at 100.00 × g and precleared by three incubations with 30 ~l of a 10% v/v suspension of protein A-CL 4B sepharose beads (Pharmacia, Uppsala, Sweden) coated with normal mouse Ig. Specific immunoprecipitation was carried out two subsequent times for 2 hr with mAb, coupled covalently to protein-A beads by means of dimethylpimelimidate-HC1 (Pierce Chemical Co., Rockford, IL). Before resuspending them in sample buffer, beads were subjected to five washes in IPB with appropriate detergent.

Gel Electrophoresis. For sodium dodecylsulphate-polyacrylamide gel electrophore- sis (SDS-PAGE), 10%-15% gradient gels were used. Samples were analyzed either under reducing conditions (5% ~-mercaptoethanol in SDS-sample buffer) or non-reducing conditions (1 mM iodoacetamide in SDS-sample buffer). Autora- diography took place at -70°C, using Kodak XAR-5 film in combination with intensifier screens (Cronex; Dupont Chemical Co., Newtown, CT).

RESULTS

BMA031 mAb exclusively detects TCR ~3 + cells in IF staining of freshly isolated T cells. In IF staining, the reactivity of BMA031 mAb was comparable to that of anti-CD3 mAbs, as tested on thymocytes and resting and activated T cells from PB [22]. However, unlike anti-CD3 mAbs, BMA031 did not react with the TCR T8 + cell line PEER [21]. This indicated that BMA031 detected a common epitope on TCR a3 and not a CD3 determinant, but such specificity could not be corrobo- rated by immunoprecipitation [21]. Since WT31, previously considered as the unique reagent for detection o fTCR a~+ cells in IF staining of intact viable cells [ 19], appeared to cross-react with TCR y8 + cells under certain conditions [31], it was useful to test the specificity of BMA031 mAb for TCR oq~ ÷ cells,

In Fig. 3, samples derived from healthy individuals and from patients suffering from different types of immunodeficiency diseases are analyzed for TCR expres- sion. A number of these patients have a high percentage of TCR y8 ÷ T cells relative to the total number of CD3 + cells [36] (~20%, Fig. 3B). As depicted in Fig. 3A, there is a very high positive correlation between WT31 and BMA031 reactivity of MNC in the 60 PB samples derived from healthy donors, as well as in the 6 BM and 84 PB samples derived from immunodeficient patients. Figure 3B shows the inverse relationship between reactivity with BMA031 and anti- TCR y8-1 mAbs of CD3 + cells in 55 PB samples derived from healthy donors,

180 J. Borst et al.

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FIGURE 3A IF staining ofTCR c~3 + and TCR 78 + cells freshly isolated from PB and BM of healthy individuals and immunodeficient patients. (A) Correlation between the percentages of BMA031 + and WT31 + cells in MNC samples from PB of 60 healthy individuals (circles), and from BM (squares) or PB (triangles) of 6 and 84 patients with immunodeficiency disease, respectively.

as well as in the 5 BM and 70 PB samples derived from immunodeficient patients. These large series of analyses demonstrate that BMA031 mAb detects a common determinant on the TCRot/~/CD3 complex, does not seem to cross-react with TCR 5/8 + cells, and is at least as useful as WT31 mAb for IF stainings of normal and patient samples.

To directly determine whether BMA031 and anti-TCR 9/8-1 mAbs stain mutu- ally exclusive cell populations, double IF stainings were performed on MNC samples isolated from PB of 10 healthy donors. For comparison, these analyses were also performed with WT31 mAb. As can be seen in Table 1, within the CD3 + population, BMA031 and WT31 detected virtually identical percentages of positive cells. A variable but significant population of anti-TCR 5/8-1 ÷ cells was present in all samples. Nei ther BMA031 nor WT31 showed reactivity with these TCR "Y3 expressing cells as determined by FACScan analysis. It can be concluded that both BMA031 and WT31 specifically detect TCR 0#3 ÷ cells in resting PB-MNC samples.

BMA031: mAb Specific for Human TCR cz/3/CD3 181

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BMA031 mAb detects exclusively TCR ~ ÷ cells in IF staining of activated T cells. In activated T-cell populations, WT31 mAb was found to react with T C R 5,8 ÷ cells as well as with T C R o#3 ÷ cells [3,31]. Therefore , BMA031 and WT31 mAbs were compared in IF staining of activated T C R 5,8 + cells (i.e., T-cell clones). Profiles obtained with three clones and the cell line PEER are shown in Fig. 1. These cells all express CD3, in association with either T C R o#3 (JS-132) or T C R 5,8 (JS-332, JS-318, PEER). T C R 5,8 can occur in two different molecular forms, with or without interchain disulphide bonds, dependent on the use of the C5,1 or the C5,2 gene segment [3,32,37]. JS-332 express C5,1, J-318 C5,2 [32].

It is clear f rom Fig. 1 that WT31 mAb stains T C R ~/3 + clones but also both types of T C R 5,8 + clones. On T C R oq3 + cells, the intensity of WT31 staining is always only slightly lower than that obtained of anti-CD3, while on T C R 5,8 + cells the intensity of WT31 staining is significantly lower than that of anti-CD3. In contrast to WT31, BMA031 mAb reacts exclusively with T C R cz/3 + clones. A very small increase in fluorescence intensity above the negative control was ob- served upon incubation of JS-318 wih BMA031 mAb. This was the most signifi- cant increase observed among at least 50 independent analyses of 20 different

182 J. Borst et al.

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FIGURE 4 Comparison performance in immunoprecipitation of BMA031 and WT31 mAbs. Clones JoBo-22 (TCR ~/3 +) and AK 4 (TCR ~8 +) were labeled by cell surface iodination and lyzed with immunoprecipitation buffer containing 0.1% Triton X-100. Lysates were precleared with normal mouse serum-coated beads, split in two, and subjected to immunoprecipitation with either BMA031 or WT31 mAb, followed by anti-CD3 mAb. Samples were analyzed by SDS-PAGE under reducing (R) and/or nonreducing (NR) conditions. Lane 1, Clone JoBo-22, normal mouse serum, NR; Lane 2, Clone JoBo-22, BMA031, NR; Lane 3, Clone JoBo-22, WT31, NR, Lane 4, Clone JoBo-22, anti-CD3, NR; Lane 5, Clone JoBo-22, normal mouse serum, R; Lane 6, Clone JoBo-22, BMA031, R; Lane 7, Clone JoBo-22, WT31, R; Lane 8, Clone JoBo-22, anti-CD3, R; Lane 9, Clone AK 4, BMA031, R; Lane 10, Clone AK 4, WT31, R; Lane 11, Clone AK 4, anti-CD3, R.

TCR ~/~ + T-cell clones and is shown primarily for that reason. Note that clone JS-318 expresses TCR yS/CD3 at very high density. We can also conclude that in IF staining of activated T cells, BMA031 rather than WT31 specifically reacts with a common epitope on the TCR ~/~/CD3 complex.

As shown in Fig. 3A, WT31 can discriminate between TCR o~3 + and TCR y~ "+ cells in PB and BM samples of healthy donors and immunodeficient patients. However , it could be envisaged that in particular patient samples T cells might be activated in vivo, and/or the TCR 5,~/CD3 complex might be differently glycosylated and/or have a different conformation, resulting in reactivity of TCR ~ ÷ cells with WT31 mAb [31]. The question was whether in such cases BMA031 mAb would still prove to be specific for TCR a/3 ÷ cells. In Fig. 2 we present a patient whose TCR T8 ~ cells, freshly isolated from PB, indeed reacted with WT31 mAb. Patient P.B. suffered from Wiskott-Aldrich syndrome, an immunode- ficiency disease. Three months after transplantation with BM of an MH C pheno-

BMA031: mAb Specific for Human TCR c~/3/CD3 183

TABLE 1 BMA031 specifically detects TCR ~/~+ cells in PB~MNC samples of 10 healthy donors

Anti-TCR~/8-1 +, WT31 +, BMA031 ÷, Donors WT31 +, CD3 + BMA031 +, CD3 ÷ CD3 + anti-TCR 5~8-1 ÷ anti-TCR "y8-1 +

H.H. 68 a 66 10.4 <0.2 <0.2 H.A. 70 67 0.8 <0.2 <0 .2 A.B. 61 59 9.3 <0.2 <0.2 D.G. 70 68 1.9 <0.2 <0 .2 P.H. 75 73 5.4 <0.2 <0.2 E.M. 71 69 9.1 <0.2 <0.2 A.Wo. 75 71 6.6 <0.2 <0 .2 R.B. 59 57 1.3 <0.2 <0.2 A.Wi. 66 61 2.6 <0.2 <0 .2 M.V. 67 66 6.1 <0.2 <0.2

Percentage positivity within the lymphocyte population as established by gating on forward and side scatter pattern in FACScan analyses.

typically compatible but genotypically unrelated donor, about 70% of CD3 ÷ lymphocytes from PB expressed TCR ~8, as determined by reactivity with anti- TCR ~/8-1 mAb. Chromosome karyotyping indicated that these cells were of recipient origin.

Double IF staining with anti-CD3 and anti-TCR ~/8-1 mAb clearly showed two CD 3 + populations, positive and negative for TCR ~8 (56.7 % versus 25.1%; Fig. 2A). Double IF staining with anti-CD3 and BMA031 mAb gave the reciprocal result, a TCR o~/3 + population of 25.7% and a negative population of 56.2% (Fig. 2B). Anti-TCR ~8-1 and BMA031 stained nonoverlapping cell populations of 25.7% and 55.8% as appears from Fig. 2C. However, WT31 mAb stained all CD3 ÷ cells (83.3%), expressing either TCR o~/~ or TCR ~/8 (Fig. 2D). This was observed repeatedly using MNC samples isolated at different time points after transplantation. Two populations with different fluorescence intensity could be discriminated. On the basis of percentages, the population with low WT31 fluo- rescence intensity (mean 45.5) would correspond to the TCR ~/8 + subset (50.7%), and the population giving WT31 staining of higher intensity (mean 91.7) to the TCR ~ + subset (25.3%). The data obtained with the sample from patient P.B. are also indicated with asterisks in Fig. 3A and B. Figures 2 and 3B clearly indicate that BMA031, rather than WT31, is the most reliable reagent to specifically detect TCR ~/3 + cells by IF analysis of normal and patient samples.

BMA031 specifically detects the TCR ~ / C D 3 complex in immunopreeipitation. The noncovalent interactions between CD3 molecules and the TCR can easily be disrupted by detergents necessary to solubilize integral membrane proteins. The most commonly used procedure for membrane protein extraction involves 1% of the closely related nonionic detergents Nonidet P-40 (NP-40) or Triton X- 100, resulting in almost complete dissociation of TCR and CD3 [38]. Lower concentrations of these detergents, such as 0.1%, are sufficient to solubilize the TCR/CD3 complex, while the intermolecular interactions are maintained. Also, detergents with other head groups, such as digitonin (at 1%) [39], can be used for extraction of the intact TCR/CD3 complex, although perhaps with lower yields.

The performance of BMA031 mAb in immunoprecipitation was first tested under conditions that allow disruption of the interactions between CD3 and TCR

184 J. Borst et al.

[1% NP-40]. In accordance with a previous report [21], BMA031 failed to specifically precipitate any structures from NP-40 lysates and in that respect behaved similarly to WT31 mAb (not shown). In previous experiments, immuno- precipitations with WT31 mAb from NP-40 lysates were only occasionally suc- cessful, and then predominantly TCR rather than CD3 components were isolated. This was the main reason for assuming that WT31 detects an epitope on the TCR rather than on the CD3 complex [19].

Next, immunoprecipitation with BMA031 and WT31 mAbs was compared under conditions that allow CD3/TCR interactions, in this case solubilization in 0.1% Triton X-100, but the same data were obtained using digitonin lysates (not shown). Figure 4 shows the performance of the two mAbs in immunoprecipitation from 0.1% Triton X-100 lysates of 125-labeled TCR ~3 + 0oBo-22) and TCR ~8 + (AK 4) T-cell clones. Clearly, WT31 mAb precipitates the intact TCR ~3/CD3 complex (lanes 3 and 7) as well as the TCR ~/~/CD3 complex (lane 10), while BMA031 exclusively precipitates the TCR ~fl/CD3 complex (compare lanes 6 and 9). Note that both BMA031 and WT31 precipitate TCR and CD3 components in seemingly equimolar amounts under these conditions of partial disruption of the TCR/CD3 complex. This supports the idea that both mAbs react with epitopes formed by TCR/CD3 interaction. Anti-CD3 mAb reacts with free as well as TCR- bound CD3 complex, resulting in a higher intensity of the CD3 bands (lanes 4, 8, 11).

DISCUSSION

BMA031 is the most useful mAb available thus far to detect viable human TCR ~3 + lymphocytes. It recognizes all TCR ~3 + cells, irrespective of variable domain diversity and does not react with TCR ~8 + cells. This has been ascertained by double IF staining of a large series of PB and BM samples from healthy individuals and immunodeficient patients with anti-CD3 and anti-TCR 78 mAb on the one hand, or anti-CD3 and BMA031 mAb on the other, as well as double IF staining of PB-MNC from 10 healthy donors with BMA031 and anti-TCR ~-1 mAbs.

BMA031 mAb can also be used for immunoprecipitation, but only under conditions where the noncovalent interactions between TCR ~/~ and the CD3 complex are preserved; i.e., in cell lysates prepared with mild detergents such as digitonin or low concentrations of the nonionic detergents Triton X-100, NP- 40, or 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate. Under such circumstances, BMA031 precipitates exclusively the TCR o~fl/CD3 complex, in contrast to WT31 mAb, which precipitates the TCR ~/~/CD3 complex as well. This result indicates that BMA031 is reactive either with a common determinant on TCR ~/~, or with a CD3 determinant, unique for TCR ~/~÷ cells. By varying the concentration of Triton X-100 from 0.02% to 0.1%, we have attempted to find conditions allowing selective isolation of TCR ~3 without CD3, but were unsuccessful. Therefore, we cannot definitively conclude that BMA031 detects an epitope on TCR ~/~ rather than on CD3. It cannot be excluded that there are epitopes on CD3 unique for TCR ~3 + cells, since CD3 components on TCR ~/~÷ cells have a different glycosylation pattern than on TCR ~/~÷ cells [40], while other biochemical differences perhaps remain to be identified.

For exactly the same reasons as outlined above for BMA031 mAb--lack of biochemical proof--it is not known whether the epitope recognized by WT31 is localized on the CD3 complex or on the TCR. Cell surface expression ofTCR ~/~ has been reconstituted in two human leukemic T-cell lines, expressing cytoplasmic TCR 3 protein and cell surface TCR ~/8 by transfection with human or murine TCR s-chain mRNA. The lines therewith gained reactivity with BMA031 and

BMA031: mAb Specific for Human TCR ~3/CD3 185

WT31 mAbs [41]. One may conclude from these results that both mAbs recognize determinants on the human TCR ~ chain. However, a recently described human leukemic T-cell line, D N D 4 1 , which expresses a/38 TCR dimer at the cell surface, does not react with either WT31 or BMA031 mAbs [42]. This implies that both determinants are not constituted by the/3 chain as such. Most likely, both mAbs recognize conformational determinants, which are dependent on the association between TCR o~/3 and CD3.

A difference between the BMA031 and WT31 epitopes is that the WT31 epitope can be unmasked on the TCR ~/8/CD3 complex by T-cell activation, sialidase treatment, or solubilization in mild detergent. Since the overall organiza- tion of the TCR o~/3/CD3 complex and the TCR yS/CD3 complex will be very similar, it is not surprising that WT31 mAb reacts with both types of complexes under certain circumstances. Another difference in reactivity of BMA031 and WT31 mAbs has been described recently [43]. A murine T-cell line was trans- fected with a cDNA clone encoding the human CD3 e chain. Transfectants showed reactivity with a number of conventional anti-CD3 mAbs, with WT31, but not with BMA031 mAb. This experiment indicates that formation of the BMA031, but not the WT31 epitope, is strictly dependent on the expression of human TCR ~/3.

Although in the great majority of cases, both in healthy individuals and in patients, WT31 reliably detects only TCR oe~ + cells in freshly isolated PB-MNC and BM samples there are situations, such as in immunodeficiency patient P.B., where TCR y~+ cells are also stained. It is not clear why WT31 mAb reacts with TCR y8 ÷ T cells directly isolated from peripheral blood of this patient. We postulate that a TCR y8 ÷ population of this patient has become activated as a result of bone marrow transplantation. We recommend the routine use ofBMA031 mAb for IF staining of not only activated T cells, but also of freshly isolated cell samples. BMA031 mAb can also be used in staining of acetone-fixed tissue sections if sensitive enzymatic detection techniques are used (T. Vroom, personal communi- cation).

ACKNOWLEDGMENTS

We thank Ms. R. Langlois-van den Berg for technical assistance; Drs. R.L.H. Bolhuis and R.J. van de Griend for supplying T-cell clones AK 4 and 1012; Drs. W.J.M. Tax, Zhang Jingwu, and W. Knapp for gifts of mAbs; M. Dessing for performing FACS analyses; and Dr. C. Figdor for critically reading the manuscript.

This work was supported by Constantijn and Christiaan Huygens Grant H 93-155 from The Netherlands Organization for Scientific Research (NWO) to J.B. and E.d.V.

REFERENCES 1. Meuer SC, Fitzgerald KA, Hussey RE, Hodgdon JC, Schlossman SF, Reinherz EL:

Clonotypic structures involved in antigen-specific human T cell function: Relationship to the T3 molecular complex. J Exp Med 157:705, 1983.

2. Brenner MB, Strominger JL, Krangel MS: The y8 T cell receptor. Adv Immunol 43:133, 1988.

3. Borst J, Van Dongen JJM, Bolhuis RLH, Peters PJ, Hailer DA, De Vries E, Van de Griend RJ: District molecular forms of human T cell receptor ~//8 detected on viable T cells by a monoclonal antibody. J Exp Med 167:1625, 1988.

4. Lanier LL, Ruitenberg J, Bolhuis RLH, Borst J, Philips JH, Testi R: Structural and serological heterogeneity of 3~/8 T cell antigen receptor expression in thymus and peripheral blood. Eur J Immunol 18:1985, 1988.

186 J. Borst et al.

5. Groh V, Porcelli S, Fabbi M, Lanier LL, Picker L, Anderson T, Warnke R, Bhan AK, Strominger JL, Brenner MB: Human lymphocytes bearing T cell receptor ~//8 are phenotypically diverse and evenly distributed throughout the lymphoid system. J Exp Med 169:1277, 1989.

6. BosJD, Teunissen MBM, Cairo I, Krieg SR, Kapsenberg ML, Das PK, BortJ: T cell receptor y~ bearing cells in normal human skin. J Invest Dermatol 94:37, 1990.

7. Vroom TM, BosJD, BorstJ: Epithelial homing of~/8 T cells? Nature 341:114, 1989.

8. Kronenberg M, Siu G, Hood LE, Shastri N: The molecular genetics of the T-cell antigen receptor and T-cell antigen recognition. Ann Rev Immunol 4:529, 1986.

9. Davis MM, Bjorkman P: T-cell antigen receptor genes and T-cell recognition. Nature 334:395, 1988.

10. Posnett DN, Bigler RD, Buskin Y, Fisher DE, Wang CY, Mayer LF, Chiorazzi N, Kunkel HG: T cell anti-idiotypic antibodies reveal differences between two human leukemias. J Exp Med 160:494, 1984.

11. Acuto O, Campen TJ, Royer HD, Hussey RE, Poole CB, Reinherz EL: Molecular analysis of T cell receptor (Ti) variable region (V) gene expression. J Exp Med 161:1326, 1985.

12. Moretta A, Pantaleo G, Lopez-Botet M, Mingari MC, Moretta L: Anti clonotypic monoclonal antibodies induce proliferation of clonotype-positive T cells in peripheral blood human T lymphocytes. J Exp Med 162:1393, 1985.

13. Borst J, Spits H, Voordouw A, De Vries E, Boylston A, De Vries JE: A family ofT- cell receptor molecules expressed on T-cell clones with different specificities for allo- major histocompatibility antigens. Hum Immunol 17:426, 1986.

14. Borst J, De Vries E, Spits H, De VriesJE, Boylston A, Matthews EA: Complexity of T cell receptor recognition sites for defined alloantigens. J Immunol 139:1952, 1987.

15. Lanier LL, RuitenbergJJ, Allison JP, Weiss A: Distinct epitopes on the T cell antigen receptor of HPB-ALL tumor cells identified by monoclonal antibodies. J Immunol 137:2286, 1986.

16. Carrel S, Isler P, Scheyer M, Vacca A, Salvi S, Giuffre L, Mach JP: Expression on human thymocytes of the idiotypic structures (Ti) from two leukemia T cell lines Jurkat and HPB-ALL. EurJ Immunol 16:649, 1986.

17. Kappler J, Kotzin B, Herron L, Gelfand EW, Bigler R, Boylston A, Carrel S, Posnett DN, Choi C, Marrack P: V/3-specific stimulation of human T cells by staphylococcal toxins. Science 244:811, 1989.

18. Tax WJM, Leeuwenberg HFM, Willems HM, Capel PJA, Koene RAP: Monoclonal antibodies reactive with the OKT3 antigen or the OKT8 antigen. In Bernard A, Boumsell L, Dausset J, Milstein C, Schlossman SF (eds): Leukocyte Typing. Berlin, Springer-Verlag, 1984, p 721.

19. Spits H, Borst J, Tax W, Capel PJA, Terhorst C, De Vries JE: Characteristics of a monoclonal antibody (WT31) that recognizes a common epitope on the human T cell receptor for antigen. J Immunol 135:1922, 1985.

20. Kurrle R, Seyfert W, Trautwein A, Seiler FR: Cellular mechanisms ofT cell activation by modulation of the T3 antigen complex. Transplant Proc 17:880, 1985.

21. Lanier LL, RuitenbergJJ, AllisonJP, Weiss A: Biochemical and flow cytometric analysis of CD3 and Ti expression on normal and malignant T-cells. In McMichael AJ, et al. (eds): Leukocyte Typing III. New York, Oxford UP, 1987, p 175.

22. Nashan B, Wonigeit K, Schwinzer R, Schlitt HJ, Kurrle R, Pichlmayr R: Fine specificity

BMA031: mAb Specific for Human TCR ~3/CD3 187

of a panel of antibodies against the TCR/CD3 complex. Transplant Proc 19:4270, 1987.

23. Kurrle R, Racenberg J, Seiler FR: BMA031-The T-cell receptor specific monoclonal antibody for immunosuppressive therapy. In TouralneJL, et al. (eds): Transplantation and Clinical Immunology, Vol 20. 1989, p 39.

24. Henry L, Tia W-T, Rittershaus C, Ko J-L, Marsh H, Ip SH: Two distinct immunogenic epitopes on the s-chain of human T cell antigen receptor. Hybridoma 8:577, 1989.

25. Brenner MB, McLean J, Scheft H, Warnke RA,Jones N, StromingerJL: Characteriza- tion and expression of the human c~ T cell receptor by using a framework monoclonal antibody. J Immunol 138:1502, 1987.

26. Fabbi M, Acuto O, Bensussan A, Poole CB, Reinherz EL: Production and characteriza- tion of antibody probes directed at constant regions of the ~ and/3 subunit of the human T cell receptor. Eur J Immunol 15:821, 1985.

27. Lew AM, Maloy WL, Koning F, Valas R, Coligan JE: Expression of the human T cell receptor as defined by anti-isotypic antibodies. J Immunol 138:807, 1987.

28. Brenner MB, McLean J, Dialynas DP, Strominger JL, Smith JA, Owen FL, Seidman JG, Ip S, Rosen F, Krangel MS: Identification of a putative second T-ceU receptor. Nature 322:145, 1986.

29. Borst J, Van de Griend RJ, Van Oostveen JW, Ang S-L, Melief CJ, Seidman JG, Bolhuis RLH: A T-cell receptor 7/CD3 complex found on cloned functional lympho- cytes. Nature 325:683, 1987.

30. Band H, Hochstenbach F, McLean J, Hata S, Krangel MS, Brenner MB: Immunochem- ical proof that a novel rearranging gene encodes the T cell receptor 8 subunit. Science 238:682, 1987.

31. Van de Griend RJ, Borst J, Tax WJM, Bolhuis RLH: Functional reactivity of WT31 monoclonal antibody with T cell receptor-~ expressing CD3 + 4 - 8 - T cells. J Immunol 140:1107, 1988.

32. Borst J, Wicherink A, Van Dongen JJM, De Vries E, Comans-Bitter WM, Wassenaar F, Van den Elsen P: Non-random expression of T cell receptor 7 and 8 variable gene segments in functional T lymphocyte clones from human peripheral blood. Eur J Immunol 19:1559, 1989.

33. Yssel H, De Vries JE, Koken M, Van Blitterswijk W, Spits H: Serum-free medium for the generation and propagation of functional human cytotoxic and helper T cell clones. J Immunol Methods 72:219, 1984.

34. Weiss A, Newton M, Crommie D: Expression of T3 in association with a molecule distinct from the T-cell antigen receptor heterodimer. Proc Natl Acad Sci USA 83:6998, 1986.

35. Spits H, Keizer G, Borst J, Terhorst C, Hekman A, De Vries JE: Characterization of monoclonal antibodies against cell surface molecules associated with cytotoxic activity of natural and activated killer cells and cloned CTL lines. Hybridoma 2:423, 1983.

36. Van Dongen JJM, Comans-Bitter WM, Friedrich W, Neijens HJ, Belohradsky BH, Kohn T, Hagemeijer A, BorstJ: Analysis of patients with DiGeorge anomaly provides evidence for extrathymic development of TCR-7~ + lymphocytes in man. Submitted for publication.

37. Van Dongen JJM, Wolvers-Tetteroo ILM, Seidman JG, Ang S-L, Van de Griend RJ, De Vries EFR, Borst J: Two types of gamma T cell receptors expressed by T cell acute lymphoblastic leukemias. Eur J Immunol 17:1719, 1987.

188 J. Borst et al.

38. BorstJ, Coligan JE, Oettgen H, Pessano S, Malin R, Terhorst C: The 6- and e-chains of the human T3/T-cell receptor complex are distinct polypeptides. Nature 312:455, 1984.

39. Oettgen HC, Pettey CL, Maloy WL, Terhorst C: A T3-1ike protein complex associated with the antigen receptor on murine T cells. Nature 320:272, 1986.

40. Krangel MS, Bierer B, Devlin P, Clabby M, Strominger JL, McLean J, Brenner MB: T3 glycoprotein is functional although structurally distinct on human T-cell receptor "/T lymphocytes. Proc Natl Acad Sci USA 84:3817, 1987.

41. De Waal Malefijt R, Alarcon B, Yssel H, Sancho J, Miyajima A, Terhorst CP, Spits H, De VriesJE: Introduction ofT cell receptor (TCR)°a cDNA has differential effects on TCR-~/8/CD3 expression by PEER and Lyon-1 cells. J Immunol 142:3634, 1989.

42. Hochstenbach F, Brenner MB: T-cell receptor 8-chain can substitute for c~ to form a 138 heterodimer. Nature 340:562-565, 1989.

43. Transy C, Moingeon PE, Marshall B, Stebbins C, Reinherz EL: Most anti-human CD3 monoclonal antibodies are directed to the CD3 ~ subunit. Eur J Immunol 19:947, 1989.