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A novel 26 kilodalton antigen expressed on the surface membrane of activated T cells
Immunobiol., vol. 200, pp. 49-61 (1999)
IDepartment of Medicine F, 2Laboratory for Immunoregulation, 3Institute of Hematology, Chaim Sheba Medical Center and Tel Aviv University, Israel; 4New York Public Health Institute and 5Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, USA
A Novel 26 Kilodalton Antigen Expressed on the Surface Membrane of Activated TCells ILAN BANK 1,2, YURI BUSHKIN4, ALEX KRITCHEVSKYS, PNINA LANGEVITZt, MAZAL BOOK 2, BORIS SHENKMAN3, RANDLE WARES, and LEONARD CHESS s
Received March 8, 1998 . Accepted in revised form June 22, 1998
Abstract We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) IBI0, raised against an activated yo T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb IBI0 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B1O+. IBI0 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) aW and yo+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were IBI0+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb IBIO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb IBIO from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb I BI a recognizes a novel «late» appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.
Abbreviations: FCS = fetal calf serum; FM = final medium; FIYC = fluoresceine isothiocyanate; LBC = lymphoblastoid B cells; IL = interleukin-2; IL-2R = IL-receptor; Ig = immunoglobulin; IV = international units; IMDM = Iscoves modified Dulbecco medium; kDa = kilodalton; MHC = major histo compatibility; mAb = monoclonal antibody; PBMC = peripheral blood mononuclear cells; pmst = phenyl methyl sulfonyl fluoride; PBS = phosphate buffered saline; R IL-2 = recombinant IL-2; SDS-PAGE = sodium dodecyl sulfate polyacrilamide gel electrophoresis; SRBC = sheep red blood cell; YCR = T cell receptor; YBS = tris buffered saline; RA = rheumatoid arthritis; VLA = very late antigen. °1999 by URBAN & FISCHER
0171-2985/99/200/01-49 $12.00/0
50 . 1. BANK et al.
Introduction T cell activation is accompanied by up-regulated expression of cell surface membrane molecules termed activation antigens (1). These include receptors for extra-cellular matrix proteins, cytokines and growth factors, as well as for cell membrane molecules (e.g. CD40L) (2-5). The activation-induced molecules are expressed on the T cell surface membrane in a temporally ordered fashion, and can be classified as «early» (e.g. CD69, CD40L and IL-2R), appearing on the T cell surface within minutes to hours, or «late» (e.g. MHC class II molecules and a1~1 and a2~1 integrins VLA-1 and 2) that become maximally expressed several days after activation (6, 7). The expression of certain activation antigens is characteristic of lymphocytic infiltrates in loci of inflammation. For example, T lymphocytes residing in the synovium in rheumatoid arthritis (RA) express «late» HLA-DR and VLA-1 antigens, as well as CD69, a very early activation antigen, whereas IL-2R, classically induced early after antigenic triggering, is expressed at very low levels on these cells (7). In this paper we describe the structure and tissue distribution of a new human T cell surface antigen, detected by mAb 1B10 (8). This mAb binds to a novel «late» 26 kDa T cell antigen that can be induced on T cells in vitro and is also expressed on T cells activated in vivo in rheumatoid synovial fluid suggesting that mAb 1B10 may be a useful new tool to evaluate T cell activation.
Materials and Methods Preparation of PBMC and T cell cultures
PBMC, sheep red blood cell (SRBC) rosetting (E+) and non-rosetting (E-) cells were obtained as previously described (9). Synovial MC were obtained by Ficoll Hypaque density centrifugation of synovial fluid obtained from three patients with inflammatory arthritis. Cells were cultured in final medium (FM) consisting of Iscoves modified Dulbecco medium (IMDM) supplemented with 10% fetal calf serum (FCS), 1% glutamine and 1% penicillin streptomycin (Sigma chemical co. St. Louis, MO, USA) solution. T cell activation was achieved by adding 5 )lg/ml of PHA (Sigma chemical co. St. Louis, MO, USA) to 5x105 E+ cells in the presence of 105 irradiated Iymphoblastoid B cells (LBC) in individual wells of 12-well tissue culture plates (Costar), each filled with 2 ml of FM. CD4+ and CDS+ TCRaW, and CD4-CDS- TCR yo+ cell lines were obtained by culturing £+ cells that had undergone negative selection by panning with the appropriate mAb (OKT4, OKTS or both mAb respectively) as previously described (10). The cells were then cloned at limiting dilutions with 100 IV/ml of recombinant IL-2 (R IL-2) (Hoffman LaRoche, Nutley, New Jersey, USA) with irradiated LBC as feeders. The CII yo clone used for the immunizations leading to isolation of the 1B10 hybridoma has been described previously (11). Preparation of 1B10 hybridoma, FACS analyses, and immuno-histochemical staining
The immunization and fusion protocols have been described elsewhere in detail (12). In brief, Balb/C mice were immunized intraperitoneally with intact CII cells, and splenocytes were fused with the non secreting myeloma P3XAg S.653 line (13). Hybridoma 1B10 secreted a mAb of the IgG1 isotype, and was submitted to the sixth international workshop on human leukocyte differentiation antigens (HLDA6), held in Kobe, Japan (14). Other mAb used in this study are: OKT4 (anti-CD4), OKT (anti-CDS), OKT3 (anti-CD3), OKT11 (anti-CD2), OKM1 (anti-
26 kDa T cell activation antigen . 51 CD11b), OKI2 (directed against HLA-DR), IB3.l (anti-VLA-1, CD49a), TCRil1 (anti-Cil), WT31 (anti-TCRa~), and anti-Tac (lL-2R) (15,16). mAb were used as diluted ascites fluid or purified mAb as indicated. Procedures for staining cells for FACS analyses were previously described; briefly, saturating concentrations of the primary murine mAb were added to a resuspended pellet of 105 cells for a 30-minute incubation period at 4°C, followed by two washes in phosphate buffered saline (PBS) pH 7.4 containing 1% FCS (16). The cells were stained for additional 30 minutes at 4 °C with goat anti-mouse (gam) immunoglobulin (lg) conjugated to fluorescein iso-thiocyanate (FITC) (gam-Ig-FITC) (Dakko, Denmark), washed twice, then analyzed on a Becton Dickinson FACS 440 or FACSCAN. For dual color staining, cells were first stained with mAb IB10 followed by gam-Ig-FITC, then with mAb directly conjugated to phycoerythrin (PE). For immuno-histology, serial 4 micron thick frozen sections were cut from cryopreserved tissue blocks, fixed in cold acetone chloroform mixture. 5 mcg/ml primary mAb were added, followed by staining with a DakoLSAB kit as previously described (17). Radio-iodination and immunoprecipitation
Aliquots of 3x10 7 cells were radio-iodinated by the lactoperoxidase method with 5 mCi of 1251 (100 mCi/ml, Amersham corp. Arlington Heights, IL, USA). To preserve inter-molecular complexes, labeled cells were then lysed with 2 ml of isotonic PBS, pH 8.3 containing 5 mM CHAPS (Sigma chemical co. St. Louis, MO, USA), 1 mM phenyl methyl sulfonyl fluoride (pmsf), 10)lM leupeptin and pepstatin A and 10 pM iodoacetamide. After depletion of free iodide by passage through a 0.2 ml column of AG1X8CT (Biorad, Richmond, CA, USA), the lysate was absorbed twice with 300 pi of gam-IgG agarose beads alone and immunoprecipitated with 60 pi of gamIgG agarose beads coated with specific mAb. The materials eluted from the beads with Laemmli sample buffer were analyzed by SDS-PAGE on vertical slabs of 11.5% acrylamide under reducing conditions (18). Treatment with N glycosidase F was performed as follows: material eluted from immune-precipitates with 50 mM Tris buffered saline (TBS) pH 7.8 containing NP-40, 0.3% SDS and 10 mM phenathroline monohydrate was digested with 2 U/ml of glycoside F (Boehringer Mannheim Biochemicals, Inidianpol, IN, USA) for 3 hours at 37°C and then analyzed by SDS-PAGE. leukemic cell lines
The leukemic T cell lines PEER (CD3+TCRyil+), Molt 4 (CDJ+CD4+CD8+CDJ+), and RPMI 8402 (CD4+CD8+CDJ+), and the myelo-monocytic U937 cell line were cultured in 75 cm 2 tissue culture flasks (Costar) at a cell density of 2-5x10 5/ml (9,12,16).
Results mAb 1B10 reacts with activated and long term cultured T lymphocytes
FACS analysis demonstrated that mAb 1B10 strongly stains the CD3+ CD4CD8- TCRyo+ clone (CII) used for immunizing mice to obtain the hybridoma (Fig. 1). In contrast, only -6% of normal peripheral blood E+ (T) cells were weakly stained. However, normal non selected E+ cells that were activated with PHA and maintained in culture for 3 weeks with R IL-2 expressed significant reactivity with mAb 1B10. Furthermore, IL-2-dependem TCR aW CD4+ (e.g. clone EKT4, Fig. 1) as well as CD8+ (e.g. clone EKT8) cells derived from normal E+ cells, and maintained in culture for >3 months, were 1B10+. T cell leukemic lines PEER, Molt 4 and RPMI 8402 were not stained by mAb 1B10 (not shown).
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Figure 1. Expression of 1BIO on T lymphocytes. TCRyl)' ClI cells, freshly isolated T cells (E+), T cells triggered with PHA then cultured for 3 weeks in vitro (E+ PHA), and cells from CD4+ (EKT4) and CD8+ (EKT8) T cell clones were stained with gam-Ig-FITC alone (column A), or with OKT3 (B), OKT8 (C), OKT4 (D), TCRl)l (E) or mAb IBIO (F) followed by gam-Ig-FITC, as described in the Methods section. 105 cells were subjected to FACS analysis: the X axis is proportional to the log fluorescence intensity (arbitrary units) and the Y axis to the number of cells.
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Relationship of 1B10 expression to T cell activation
in vitro and in vivo
Collectively, the lack of significant reactivity of most resting T cells, and the reactivity with PHA activated T cells and T cell clones, suggested that expression of the antigen recognized by mAb IBl on T cells occurs after their activation. To evaluate the kinetics of induction of the IBI0 antigen in relation to an early activation antigen, resting T lymphocytes were activated with PHA. As expected, high levels of IL-2R were induced on the cells within 24 hours of culture (not shown). In contrast, reactivity with mAb IBI0 was negligible after this brief period of in vitro culture. However, as depicted in Figure 2, after 96 hours in culture in the presence of PHA, 34% of the non-selected T cells, 55% of the CD+4 T cells and 23% of the CD8+ cells become IBI0+, suggesting that the antigen detected by mAb 1BlOis induced as a «late» T cell activation antigen. To further correlate IBI0 expression with IL-2R and HLA-DR, classical activation antigens of T cells, dual staining of T cells activated for 6 days with recombinant IL-2 was performed. As shown in Table 1, although most 1BI0+ cells coexpressed IL-2R and HLA-DR, a smaller proportion did not express these activation markers. In addition, we compared the levels of expression of 1B10 and those of two other late T cell activation markers, HLA-DR and VLA-l in T cell clones that were cultured with IL-2 for prolonged periods (>3 months). The CD4+ clone showing in Figure 3 expressed high levels of IBI0 antigen but low levels of HLA-DR and VLA-1. The CD8+ clone represented in Figure 3, on the other hand, expressed high levels of all three activation antigens. Clone PLT4 expressed intermediate levels of late activation antigens HLA-DR, VLA-l and IBI0 and low levels of IL-2R, clone DMT4 expressed high levels of HLA-DR and IL-2R with low levels of VLA-l and IBI0, and clone DM12 did not express HLA-DR at all, but did express low but significant levels of IBI0 and VLA-l (Table 3). The lEg yo clone expressed high level VLA-l and a significant but 60 r - - - - - - - - - - - - - - - - ,
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Figure 2. Activation-induced expression of IBID on resting T cells. Unfractionated E+ cells, or purified DC4+ or CD8+ sub-populations from the same individual, were triggered with PHA, then stained with mAb lBlO or a negative control antibody (OKT6) after 96,120 and 144 hours in culte (X axis). The Y axis shows the percent of cells stained by mAb IBID with a fluorescence intensity exceeding that of 99.5% of the cells in the corresponding negative control.
54 . I. BANK et al. CD4+ clone 175
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Log fluorescence intensity Figure 3. Comparative expression of 3 late T cell activation antigens. FACS analyses of a CD4+ and a CD8+ T cell clone obtained from the PB of a normal individual as described in materials and methods, following staining with gam-Ig-FITC alone (indicated in the figure as control), or with mAbs OKIl2 (anti HLA-DR), IB3.1 (anti VLA-I) and IBID. The X axis is proportional to log fluorescence intensity, and the Y axis to the cell number.
26 kDa T cell activation antigen . 55 Table 1. Co-expression of 1B10, HLA-DR and IL-2R.
HLA-DR+ HLA-DRIL-2R+ IL-2R-
1B10+
1B1O-
9.5
18.3 67.5 11.7 78
4.7
7.5 2.8
Percentage of T cells co-expressing the 1B10 antigen, IL-2R and HLA-DR after 6 days of activation of PBMC with recombinant IL-2 (100 u/ml). Staining was performed with mAb 1B10 followed by gam-Ig-FITC, followed by PE-conjugated mAb to IL-2R and HLA-DR (Becton Dickinson).
Table 2. Expression of 1B10 antigen relative to VLA-1, HLA-DR and IL-2R. antigen/clone
negative
CD3
CD4
CD8
VLA-1
HLA-DR
IL-2R
1B10
PLT4';DMT4';DM1Y IE9(y() clone)#
4 5 4 54
107 241 195 181
130 241 ND 65
4 6 ND 63
39 7 30 151
40 306 2.5 ND
7 89 ND ND
41 8 17 81
Logarithmic of mean fluorescence intensity (arbitrary units) after staining clones with mAb to the indicated antigens, then with gam-Ig-FITC. Negative: gam-Ig-FITC with no primary mAb. Analyzed on FACSCAN';- or FACS 440# Table 3. Tissue
Positive structures
Thymus Lymph node
Hassals corpuscles, dendritic cells in medulla and cortex Some germinal center macrophages, some follicular dendritic cells Macrophages and dendritic cells of white and red pulp Hepatocytes, sinusoids Proximal tubules Basal layer fibroblasts Rare alveolar lining cells Glands Fibroblasts Blood vessels Lining of cystic spaces Seminiferous tubules Basal epithelium fibroblasts Glands Glands Deep glands macrophages
Spleen Liver Kidney Skin Lung Uterus Heart Brain Ovary Testis Oesophagus Prostate Colon Stomach
Degree of positivity + +
+
+/++ +/-
+ + ++ +/++ +
++
+/-
+
++
+ + +
Samples from frozen sections of the indicated tissues were stained with mAb 1B10 as described in Materials and Methods. The intensity of staining of the positive structures in each section was graded as very strong (++), strong (+) or weak (+/-).
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Figure 4. 1BIO expression on synovial T lymphocytes. SMC were stained with gam-Ig-FITC alone (F) or the mAb indicated in the figure followed by gam-Ig-FITC, and analyzed by FACS. Representative of samples drawn from 3 patients with active rheumatoid arthritis.
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lower level of 1B1o. Finally, FACS analysis of T cells isolated from the synovial fluid of 3 patients with active RA were performed (7, 13). As can be seen in Figure 4, activated CD3+CD4+ synovial T Cells (HLA-DR+) were also lBl0+. Taken together, the data indicate that lBl0 is expressed in vitro on activated T cells independently of HLA-DR and VLA-l, and can be found on T cells activated in vivo. 1B10 reactivity in non-T cells
Immuno-histochemical studies revealed that mAb lBl0 stained dendritic cells and macrophages in thymus, lymph nodes and spleen, but did not stain lymphocytes in these lymphoid tissues (Table 3). Non-lymphoid cells stained prominently by mAb 1B10 included secretory epithelia of glands in breast, prostate and kidney. LBC were in general either negative or weakly positive. In addition, as shown in Figure 5, FACS analysis revealed that mAb lBl0 strongly stained the U937 myelo-monocytic cell line.
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Figure S. U937 cells express IBIO antigen. Cultured U937 cells were stained with gam-Ig-FITC alone (F), or with anti-CD2 mAb OKT11, or with mAb IBIO followed by gam-Ig-FITC and subjected to FACS analyses.
Immuno-precipitation of the 1B10 antigen
We next identified the antigen recognized by mAb lBl0. SDS-PAGE analysis of surface molecules immuno-precipitated by mAb lBl0 from radio-iodinated surface membranes of the ClI y'6 T cell line revealed a 26 kDa glycoprotein which migrated as a 22 kDa peptide following treatment with N glycosidase F (Fig. 6A, lanes 2 and 4) (11, 19). In contrast, as a control, anti-CD3 mAb immunoprecipitated CD3 glycoproteins associated with the y'6 TCR from the ClI cell surface membrane, that migrated as 18 kDa peptides after deglycosylation (Fig. 6A, lanes 1 and 3) (10). In a parallel experiment, mAb lBl0 precipitated a 29 kDa glycoprotein from U937 lysates (Fig. 6B, lane 2), which migrated as a 22 kDa peptide after deglycosylation (Fig. 6B, lane 3). Isotype control mAb did not immunoprecipitate any material in these experiments (not shown).
58 . 1. BANK et al.
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Discussion We have described the tissue distribution and initial biochemical characterization of a novel glycoprotein expressed on T cells activated in vitro, as well as in vivo, while being expressed on only a small fraction of resting T lymphocytes in normal peripheral blood and lymphoid tissues. Interestingly, this T cell activation antigen is also expressed on tissue resident macrophages and dendritic cells and is quite prominent in epithelial cells suggesting that the novel 1B10 antigen is a non lymphoid-specific surface antigen that becomes up-regulated on activated T cells. The immunoprecipitation experiments demonstrated that the Mr of the 1B10 antigen ranges from 26-29 kDa due to differential glycosylation of a 22 kDa peptide (Fig. 6). Previously described T cell activation antigens of similar Mr include the CD69 and CD9 molecules (19, 20). In contrast to CD69 which is expressed within minutes after T cell activation, prior to IL-2R, the 1B10 antigen is increasingly expressed on activated T cells only after high levels of IL-2R are achieved on the cell surface (Fig. 2 and ref. 20). CD69 consists of disulfide linked 28/32 kDa subunits and is strongly expressed on immature thymocytes, whereas the 1B10 antigen is expressed as a single chain glycoprotein in the cell surface membrane and is not detected in thymocytes (Fig. 6 and Table 1). Taken together these data indicate that 1B10 antigen is clearly distinct from CD69. Since 1B10 antigen neither strains nor activates human platelets (data not shown), this antigen also differs from CD9 which is expressed by platelets and activates them (21, 22). Further analyses of the structure of the 1B10 glycoprotein will be required to determine whether it bears homology to other known leukocyte CD antigens. Preliminary studies employing mAb 1B10 as an activating or blocking agent in vitro have failed to reveal an effect on T cell-mediated cytotoxicity or allogeneic recognition, suggesting that the 1B10 molecule is not involved in these classical T cell functions (data not shown). The constitutive expression of 1B10 on tissue resident macrophages and epithelial cells (Table 1) suggests it may function as an adhesion molecules. 1B10 expression, irrespective of it's function, may provide an additional useful marker to detect and to define activated lymphocytes in vivo since 1B10 is maximally expressed at a relatively late stage after activation, after induction of IL-2R, and it's expression is usually maintained on
•
Figure 6. SDS-PAGE analysis of molecules precipitated by mAb IBIO from radio iodinated lysates ofT cells and U937 cells. A. T cell (clone Cn) lysates were precipitated with mAb OKT3 (lanes 1 and 3) or mAb IBIO (lanes 2 and 4) before (lanes 1 and 2) and after (lanes 3 and 4) digestion with N glycosidase F. B. U937 cells precipitated with beads alone (lane 1) or with mAb IBIO, before (lane 2), and after (lane 3) digestion with N glycosidase F respectively. The open arrows indicate the positions of fully glycosylated CD3y (lane IA) and IBIO (lanes 2A and B) molecules precipitated from the lysates, whereas blackened arrows indicate deglycosylated forms of these molecules. The 43 kDa protein co-precipitated by OKT3 (Fig. 6A, lane 1) represents the yo TCR. The nature of the contaminating band migrating at 40 kDa (Fig. 6B, lanes 1-3) is unknown.
60 . 1. BANK et al.
long term previously activated T cells (Fig. 1-3). Furthermore, the levels of 1B10 are independent of VLA-1 and HLA-DR, two classical late activation markers, which may facilitate the detection of VLA-1 low and HLA-DR low previously activated T cell clones in vivo (Fig. 4). Interestingly, in a preliminary study we determined that the level of expression of 1B10 antigen in the PBMC of patients with RA is diminished during therapy with minocycline along with clinical improvement (8). It will be of interest to determine whether the level of expression of 1B10 contributes to T cell migration or localization to the joint space, to the presumed pathogenicity of the cells in the synovium, or to other specific functions of activated T cells in vivo.
References 1. COTNER, T.,]. M. WILLIAMS, L. CHRISTENSON, H. M. SHAPIRO, T. B. STROM, and J. STROMINGER. 1983. Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content.]. Exp. Med. 157: 461. 2. HEMLER, M. E., and]. G. JACOBSON. 1987. Cell matrix adhesion-related proteins VLA-l and VLA-2: regulation of expression on T cells.]. Immunol. 138: 2941. 3. HEMLER, M. E., ]. G. JACOBSON, M. B. BRENNER, D. MANN, and]. L. STROMINGER. 1985. VLA-l: a T cell surface antigen which defines a novel late stage of human T cell activation. Eur. ]. Immunol. 15: 502. 4. LEONARD, W. J.,]. M. DEPPER, T. UCHIYAMA, K. A. SMITH, T. A. WALDMANN, and W. C. GREENE. 1982. A monoclonal antibody appears to recognize the receptor for human T cell growth factor: partial characterization of the receptor. Nature (Lond.) 300: 267. 5. LEDERMAN, S., M. ]. YELLIN, A. M. CLEARY, S. M. FORTUNE, and L. CHESS. 1994. The understanding of contact dependent T-cell helper function in molecular, cellular and physiological detail. Res. Immunol. 145: 215. 6 MASUYAMA, J. 1.,]. S. BERMAN, W. W. CRUISHANK, C. MORIMOTO, and D. CENTER. 1992. Evidence for recent as well as long term activation of T cell migrating through endothelial cell monolayers in vitro.]. Immunol. 148: 1367. 7. IANNONE, E, V. M. CORRIGALL, G. H. KINGSLEY, and G. S. PANAYI. 199 Evidence for the continuous recruitment and activation of T cells into the joints of patients with rheumatoid arthritis. Eur. ]. Immunol. 24: 2706. 8. LANGEVITZ, P., 1. BANK, D. ZENNER, M. BOOK, and M. PRAS. 1992. Treatment of resistant rheumatoid arthritis with minocycline: an open study.]. Rheumatol. 19: 1502. 9. BANK, 1., and L. CHESS. 1985. Perturbation of the T4 molecule transmits a negative signal to T cells.]. Exp. Med. 162: 1294. T cells from a 10. BANK 1., A. TANAY, A. MIGDAL, M. BOOK, and A. LIVNEH. 1995. V"fJ-V02+ patient with Felty syndrome that exhibit aberrant responses to triggering of the CD3 molecule can regulate immunoglobulin secretion by B cells. Clin. Immunol. and Immunopathol. 74: 162. 11. BANK, 1., R. A. DEPINHO, M. B. BRENNER, ]. CASSIMERIS, E ALT, and L. CHESS. 1986. A novel heterodimer on the surface of immature thymocytes. Nature 322: 179. 12. BANK, 1., M. HEMLER, M. B. BRENNER, D. COHEN, V. LEVY,]. BELKO, C. CROUSE, and L. CHESS. 1989. A novel monoclonal antibody, IB3.1, binds to a new epitope of the VLA-l molecule. Cell Immunol. 122: 416. 13. KRICHEVSKY, A., E. G. ARMSTRONG, ]. SCHLATTERER, S. BIRKEN, ]. O'CONNOR, K. BIKEL, S., SILVERBERG,]. W. LUSTBADER, and R. E. CANFIELD. 1988. Preparation and characterization of antibodies to the urinary fragment of human chorionic gonadotrophin ~o subunit. Endocrinology 123: 584.
yo
26 kDa T cell activation antigen . 61 14. KISHIMOTO, T, H. KIKUTANI, A. E. G. VON OEM BORNE, et al. 1997. Leukocyte typing VI. Garland Publishing Inc. New York & London, pp. 114. 15. BANK, I., D. ROTH, M. BOOK, R. BLOCK, P. LANGEVITZ, M. EHRENFELD, and M. PRAS. 1991. Expression and functions of very late antigen 1 in inflammatory joint diseases. J. Clin. Immunol. 11: 29. 16. BANK, I., M. BOOK, and R. WARE. 1994. Functional role of VLA-1 (CD49a) in adhesion, cation dependent spreading, and activation of cultured human T lymphocytes. Cell Immunol. 156: 424. 17. BANK, 1.., M. BOOK, M. HUSZAR, Y. BARAM, I. SCHNIRER, and H. BRENNER. 1993. V'62' T lymphocytes are cytotoxic to the MCF7 breast carcinoma cell line and can be detected among the y'6 T cells that infiltrate breast carcinoma. Clin. Immunol. and Immunopathol. 67: 17. 18. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage n. Nature (Lond.) 227: 680. 19. OKADA, A., I. BANK, L. ROGOZINSKY, Y. TAKIHARA, T M. MAK, L. CHESS, and E W. ALT. 1988. Structure of the y'6 T cell receptor of a human thymocyte clone. J. Exp. Med. 168: 1481. 20. CEBRIAN, M., E. YAGUE, M, RINCON, M. LOPEZ BOTET, M. O. DE LANDAZURI, and E J. SANCHEZ MADRID. 1988. Triggering of T cell proliferation through AIM, an activation inducer molecule expressed on activated human lymphocytes. J. Exp. Med. 168: 162. 21. JENNINGS, L. K., J. T CROSSNO, and M. M. WHITE. 1995. CD9 cluster workshop report: cell surface binding and functional analysis. Leuocyte typing V. White cell differentiation antigens. Editors, SCHLOSMAN, S. E, et al. Oxford University Press, pp. 1249. 22. KROLL, M. H., M. E. MENDELSOHN, J. L. MILLER BALLEN, K. K. J. K. HRBOLlCH, and A. I. SCHAFER. 1992. Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIa and extracellular calcium. Biochem. Biophys. Acta 1137: 248. Dr. I. BANK, Dept. of Medicine F, Chaim Sheba Medical Center, Tel Hashomer 52621 Israel. Fax.: ++972 353 024 90; Tel.: ++972 354 099 69.
IDepartment of Medicine F, 2Laboratory for Immunoregulation, 3Institute of Hematology, Chaim Sheba Medical Center and Tel Aviv University, Israel; 4New York Public Health Institute and 5Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, USA
A Novel 26 Kilodalton Antigen Expressed on the Surface Membrane of Activated TCells ILAN BANK 1,2, YURI BUSHKIN4, ALEX KRITCHEVSKYS, PNINA LANGEVITZt, MAZAL BOOK 2, BORIS SHENKMAN3, RANDLE WARES, and LEONARD CHESS s
Received March 8, 1998 . Accepted in revised form June 22, 1998
Abstract We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) IBI0, raised against an activated yo T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb IBI0 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B1O+. IBI0 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) aW and yo+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were IBI0+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb IBIO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb IBIO from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb I BI a recognizes a novel «late» appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.
Abbreviations: FCS = fetal calf serum; FM = final medium; FIYC = fluoresceine isothiocyanate; LBC = lymphoblastoid B cells; IL = interleukin-2; IL-2R = IL-receptor; Ig = immunoglobulin; IV = international units; IMDM = Iscoves modified Dulbecco medium; kDa = kilodalton; MHC = major histo compatibility; mAb = monoclonal antibody; PBMC = peripheral blood mononuclear cells; pmst = phenyl methyl sulfonyl fluoride; PBS = phosphate buffered saline; R IL-2 = recombinant IL-2; SDS-PAGE = sodium dodecyl sulfate polyacrilamide gel electrophoresis; SRBC = sheep red blood cell; YCR = T cell receptor; YBS = tris buffered saline; RA = rheumatoid arthritis; VLA = very late antigen. °1999 by URBAN & FISCHER
0171-2985/99/200/01-49 $12.00/0
50 . 1. BANK et al.
Introduction T cell activation is accompanied by up-regulated expression of cell surface membrane molecules termed activation antigens (1). These include receptors for extra-cellular matrix proteins, cytokines and growth factors, as well as for cell membrane molecules (e.g. CD40L) (2-5). The activation-induced molecules are expressed on the T cell surface membrane in a temporally ordered fashion, and can be classified as «early» (e.g. CD69, CD40L and IL-2R), appearing on the T cell surface within minutes to hours, or «late» (e.g. MHC class II molecules and a1~1 and a2~1 integrins VLA-1 and 2) that become maximally expressed several days after activation (6, 7). The expression of certain activation antigens is characteristic of lymphocytic infiltrates in loci of inflammation. For example, T lymphocytes residing in the synovium in rheumatoid arthritis (RA) express «late» HLA-DR and VLA-1 antigens, as well as CD69, a very early activation antigen, whereas IL-2R, classically induced early after antigenic triggering, is expressed at very low levels on these cells (7). In this paper we describe the structure and tissue distribution of a new human T cell surface antigen, detected by mAb 1B10 (8). This mAb binds to a novel «late» 26 kDa T cell antigen that can be induced on T cells in vitro and is also expressed on T cells activated in vivo in rheumatoid synovial fluid suggesting that mAb 1B10 may be a useful new tool to evaluate T cell activation.
Materials and Methods Preparation of PBMC and T cell cultures
PBMC, sheep red blood cell (SRBC) rosetting (E+) and non-rosetting (E-) cells were obtained as previously described (9). Synovial MC were obtained by Ficoll Hypaque density centrifugation of synovial fluid obtained from three patients with inflammatory arthritis. Cells were cultured in final medium (FM) consisting of Iscoves modified Dulbecco medium (IMDM) supplemented with 10% fetal calf serum (FCS), 1% glutamine and 1% penicillin streptomycin (Sigma chemical co. St. Louis, MO, USA) solution. T cell activation was achieved by adding 5 )lg/ml of PHA (Sigma chemical co. St. Louis, MO, USA) to 5x105 E+ cells in the presence of 105 irradiated Iymphoblastoid B cells (LBC) in individual wells of 12-well tissue culture plates (Costar), each filled with 2 ml of FM. CD4+ and CDS+ TCRaW, and CD4-CDS- TCR yo+ cell lines were obtained by culturing £+ cells that had undergone negative selection by panning with the appropriate mAb (OKT4, OKTS or both mAb respectively) as previously described (10). The cells were then cloned at limiting dilutions with 100 IV/ml of recombinant IL-2 (R IL-2) (Hoffman LaRoche, Nutley, New Jersey, USA) with irradiated LBC as feeders. The CII yo clone used for the immunizations leading to isolation of the 1B10 hybridoma has been described previously (11). Preparation of 1B10 hybridoma, FACS analyses, and immuno-histochemical staining
The immunization and fusion protocols have been described elsewhere in detail (12). In brief, Balb/C mice were immunized intraperitoneally with intact CII cells, and splenocytes were fused with the non secreting myeloma P3XAg S.653 line (13). Hybridoma 1B10 secreted a mAb of the IgG1 isotype, and was submitted to the sixth international workshop on human leukocyte differentiation antigens (HLDA6), held in Kobe, Japan (14). Other mAb used in this study are: OKT4 (anti-CD4), OKT (anti-CDS), OKT3 (anti-CD3), OKT11 (anti-CD2), OKM1 (anti-
26 kDa T cell activation antigen . 51 CD11b), OKI2 (directed against HLA-DR), IB3.l (anti-VLA-1, CD49a), TCRil1 (anti-Cil), WT31 (anti-TCRa~), and anti-Tac (lL-2R) (15,16). mAb were used as diluted ascites fluid or purified mAb as indicated. Procedures for staining cells for FACS analyses were previously described; briefly, saturating concentrations of the primary murine mAb were added to a resuspended pellet of 105 cells for a 30-minute incubation period at 4°C, followed by two washes in phosphate buffered saline (PBS) pH 7.4 containing 1% FCS (16). The cells were stained for additional 30 minutes at 4 °C with goat anti-mouse (gam) immunoglobulin (lg) conjugated to fluorescein iso-thiocyanate (FITC) (gam-Ig-FITC) (Dakko, Denmark), washed twice, then analyzed on a Becton Dickinson FACS 440 or FACSCAN. For dual color staining, cells were first stained with mAb IB10 followed by gam-Ig-FITC, then with mAb directly conjugated to phycoerythrin (PE). For immuno-histology, serial 4 micron thick frozen sections were cut from cryopreserved tissue blocks, fixed in cold acetone chloroform mixture. 5 mcg/ml primary mAb were added, followed by staining with a DakoLSAB kit as previously described (17). Radio-iodination and immunoprecipitation
Aliquots of 3x10 7 cells were radio-iodinated by the lactoperoxidase method with 5 mCi of 1251 (100 mCi/ml, Amersham corp. Arlington Heights, IL, USA). To preserve inter-molecular complexes, labeled cells were then lysed with 2 ml of isotonic PBS, pH 8.3 containing 5 mM CHAPS (Sigma chemical co. St. Louis, MO, USA), 1 mM phenyl methyl sulfonyl fluoride (pmsf), 10)lM leupeptin and pepstatin A and 10 pM iodoacetamide. After depletion of free iodide by passage through a 0.2 ml column of AG1X8CT (Biorad, Richmond, CA, USA), the lysate was absorbed twice with 300 pi of gam-IgG agarose beads alone and immunoprecipitated with 60 pi of gamIgG agarose beads coated with specific mAb. The materials eluted from the beads with Laemmli sample buffer were analyzed by SDS-PAGE on vertical slabs of 11.5% acrylamide under reducing conditions (18). Treatment with N glycosidase F was performed as follows: material eluted from immune-precipitates with 50 mM Tris buffered saline (TBS) pH 7.8 containing NP-40, 0.3% SDS and 10 mM phenathroline monohydrate was digested with 2 U/ml of glycoside F (Boehringer Mannheim Biochemicals, Inidianpol, IN, USA) for 3 hours at 37°C and then analyzed by SDS-PAGE. leukemic cell lines
The leukemic T cell lines PEER (CD3+TCRyil+), Molt 4 (CDJ+CD4+CD8+CDJ+), and RPMI 8402 (CD4+CD8+CDJ+), and the myelo-monocytic U937 cell line were cultured in 75 cm 2 tissue culture flasks (Costar) at a cell density of 2-5x10 5/ml (9,12,16).
Results mAb 1B10 reacts with activated and long term cultured T lymphocytes
FACS analysis demonstrated that mAb 1B10 strongly stains the CD3+ CD4CD8- TCRyo+ clone (CII) used for immunizing mice to obtain the hybridoma (Fig. 1). In contrast, only -6% of normal peripheral blood E+ (T) cells were weakly stained. However, normal non selected E+ cells that were activated with PHA and maintained in culture for 3 weeks with R IL-2 expressed significant reactivity with mAb 1B10. Furthermore, IL-2-dependem TCR aW CD4+ (e.g. clone EKT4, Fig. 1) as well as CD8+ (e.g. clone EKT8) cells derived from normal E+ cells, and maintained in culture for >3 months, were 1B10+. T cell leukemic lines PEER, Molt 4 and RPMI 8402 were not stained by mAb 1B10 (not shown).
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Figure 1. Expression of 1BIO on T lymphocytes. TCRyl)' ClI cells, freshly isolated T cells (E+), T cells triggered with PHA then cultured for 3 weeks in vitro (E+ PHA), and cells from CD4+ (EKT4) and CD8+ (EKT8) T cell clones were stained with gam-Ig-FITC alone (column A), or with OKT3 (B), OKT8 (C), OKT4 (D), TCRl)l (E) or mAb IBIO (F) followed by gam-Ig-FITC, as described in the Methods section. 105 cells were subjected to FACS analysis: the X axis is proportional to the log fluorescence intensity (arbitrary units) and the Y axis to the number of cells.
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Relationship of 1B10 expression to T cell activation
in vitro and in vivo
Collectively, the lack of significant reactivity of most resting T cells, and the reactivity with PHA activated T cells and T cell clones, suggested that expression of the antigen recognized by mAb IBl on T cells occurs after their activation. To evaluate the kinetics of induction of the IBI0 antigen in relation to an early activation antigen, resting T lymphocytes were activated with PHA. As expected, high levels of IL-2R were induced on the cells within 24 hours of culture (not shown). In contrast, reactivity with mAb IBI0 was negligible after this brief period of in vitro culture. However, as depicted in Figure 2, after 96 hours in culture in the presence of PHA, 34% of the non-selected T cells, 55% of the CD+4 T cells and 23% of the CD8+ cells become IBI0+, suggesting that the antigen detected by mAb 1BlOis induced as a «late» T cell activation antigen. To further correlate IBI0 expression with IL-2R and HLA-DR, classical activation antigens of T cells, dual staining of T cells activated for 6 days with recombinant IL-2 was performed. As shown in Table 1, although most 1BI0+ cells coexpressed IL-2R and HLA-DR, a smaller proportion did not express these activation markers. In addition, we compared the levels of expression of 1B10 and those of two other late T cell activation markers, HLA-DR and VLA-l in T cell clones that were cultured with IL-2 for prolonged periods (>3 months). The CD4+ clone showing in Figure 3 expressed high levels of IBI0 antigen but low levels of HLA-DR and VLA-1. The CD8+ clone represented in Figure 3, on the other hand, expressed high levels of all three activation antigens. Clone PLT4 expressed intermediate levels of late activation antigens HLA-DR, VLA-l and IBI0 and low levels of IL-2R, clone DMT4 expressed high levels of HLA-DR and IL-2R with low levels of VLA-l and IBI0, and clone DM12 did not express HLA-DR at all, but did express low but significant levels of IBI0 and VLA-l (Table 3). The lEg yo clone expressed high level VLA-l and a significant but 60 r - - - - - - - - - - - - - - - - ,
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Figure 2. Activation-induced expression of IBID on resting T cells. Unfractionated E+ cells, or purified DC4+ or CD8+ sub-populations from the same individual, were triggered with PHA, then stained with mAb lBlO or a negative control antibody (OKT6) after 96,120 and 144 hours in culte (X axis). The Y axis shows the percent of cells stained by mAb IBID with a fluorescence intensity exceeding that of 99.5% of the cells in the corresponding negative control.
54 . I. BANK et al. CD4+ clone 175
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Log fluorescence intensity Figure 3. Comparative expression of 3 late T cell activation antigens. FACS analyses of a CD4+ and a CD8+ T cell clone obtained from the PB of a normal individual as described in materials and methods, following staining with gam-Ig-FITC alone (indicated in the figure as control), or with mAbs OKIl2 (anti HLA-DR), IB3.1 (anti VLA-I) and IBID. The X axis is proportional to log fluorescence intensity, and the Y axis to the cell number.
26 kDa T cell activation antigen . 55 Table 1. Co-expression of 1B10, HLA-DR and IL-2R.
HLA-DR+ HLA-DRIL-2R+ IL-2R-
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Percentage of T cells co-expressing the 1B10 antigen, IL-2R and HLA-DR after 6 days of activation of PBMC with recombinant IL-2 (100 u/ml). Staining was performed with mAb 1B10 followed by gam-Ig-FITC, followed by PE-conjugated mAb to IL-2R and HLA-DR (Becton Dickinson).
Table 2. Expression of 1B10 antigen relative to VLA-1, HLA-DR and IL-2R. antigen/clone
negative
CD3
CD4
CD8
VLA-1
HLA-DR
IL-2R
1B10
PLT4';DMT4';DM1Y IE9(y() clone)#
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107 241 195 181
130 241 ND 65
4 6 ND 63
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40 306 2.5 ND
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41 8 17 81
Logarithmic of mean fluorescence intensity (arbitrary units) after staining clones with mAb to the indicated antigens, then with gam-Ig-FITC. Negative: gam-Ig-FITC with no primary mAb. Analyzed on FACSCAN';- or FACS 440# Table 3. Tissue
Positive structures
Thymus Lymph node
Hassals corpuscles, dendritic cells in medulla and cortex Some germinal center macrophages, some follicular dendritic cells Macrophages and dendritic cells of white and red pulp Hepatocytes, sinusoids Proximal tubules Basal layer fibroblasts Rare alveolar lining cells Glands Fibroblasts Blood vessels Lining of cystic spaces Seminiferous tubules Basal epithelium fibroblasts Glands Glands Deep glands macrophages
Spleen Liver Kidney Skin Lung Uterus Heart Brain Ovary Testis Oesophagus Prostate Colon Stomach
Degree of positivity + +
+
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+ + ++ +/++ +
++
+/-
+
++
+ + +
Samples from frozen sections of the indicated tissues were stained with mAb 1B10 as described in Materials and Methods. The intensity of staining of the positive structures in each section was graded as very strong (++), strong (+) or weak (+/-).
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Figure 4. 1BIO expression on synovial T lymphocytes. SMC were stained with gam-Ig-FITC alone (F) or the mAb indicated in the figure followed by gam-Ig-FITC, and analyzed by FACS. Representative of samples drawn from 3 patients with active rheumatoid arthritis.
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lower level of 1B1o. Finally, FACS analysis of T cells isolated from the synovial fluid of 3 patients with active RA were performed (7, 13). As can be seen in Figure 4, activated CD3+CD4+ synovial T Cells (HLA-DR+) were also lBl0+. Taken together, the data indicate that lBl0 is expressed in vitro on activated T cells independently of HLA-DR and VLA-l, and can be found on T cells activated in vivo. 1B10 reactivity in non-T cells
Immuno-histochemical studies revealed that mAb lBl0 stained dendritic cells and macrophages in thymus, lymph nodes and spleen, but did not stain lymphocytes in these lymphoid tissues (Table 3). Non-lymphoid cells stained prominently by mAb 1B10 included secretory epithelia of glands in breast, prostate and kidney. LBC were in general either negative or weakly positive. In addition, as shown in Figure 5, FACS analysis revealed that mAb lBl0 strongly stained the U937 myelo-monocytic cell line.
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Immuno-precipitation of the 1B10 antigen
We next identified the antigen recognized by mAb lBl0. SDS-PAGE analysis of surface molecules immuno-precipitated by mAb lBl0 from radio-iodinated surface membranes of the ClI y'6 T cell line revealed a 26 kDa glycoprotein which migrated as a 22 kDa peptide following treatment with N glycosidase F (Fig. 6A, lanes 2 and 4) (11, 19). In contrast, as a control, anti-CD3 mAb immunoprecipitated CD3 glycoproteins associated with the y'6 TCR from the ClI cell surface membrane, that migrated as 18 kDa peptides after deglycosylation (Fig. 6A, lanes 1 and 3) (10). In a parallel experiment, mAb lBl0 precipitated a 29 kDa glycoprotein from U937 lysates (Fig. 6B, lane 2), which migrated as a 22 kDa peptide after deglycosylation (Fig. 6B, lane 3). Isotype control mAb did not immunoprecipitate any material in these experiments (not shown).
58 . 1. BANK et al.
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Discussion We have described the tissue distribution and initial biochemical characterization of a novel glycoprotein expressed on T cells activated in vitro, as well as in vivo, while being expressed on only a small fraction of resting T lymphocytes in normal peripheral blood and lymphoid tissues. Interestingly, this T cell activation antigen is also expressed on tissue resident macrophages and dendritic cells and is quite prominent in epithelial cells suggesting that the novel 1B10 antigen is a non lymphoid-specific surface antigen that becomes up-regulated on activated T cells. The immunoprecipitation experiments demonstrated that the Mr of the 1B10 antigen ranges from 26-29 kDa due to differential glycosylation of a 22 kDa peptide (Fig. 6). Previously described T cell activation antigens of similar Mr include the CD69 and CD9 molecules (19, 20). In contrast to CD69 which is expressed within minutes after T cell activation, prior to IL-2R, the 1B10 antigen is increasingly expressed on activated T cells only after high levels of IL-2R are achieved on the cell surface (Fig. 2 and ref. 20). CD69 consists of disulfide linked 28/32 kDa subunits and is strongly expressed on immature thymocytes, whereas the 1B10 antigen is expressed as a single chain glycoprotein in the cell surface membrane and is not detected in thymocytes (Fig. 6 and Table 1). Taken together these data indicate that 1B10 antigen is clearly distinct from CD69. Since 1B10 antigen neither strains nor activates human platelets (data not shown), this antigen also differs from CD9 which is expressed by platelets and activates them (21, 22). Further analyses of the structure of the 1B10 glycoprotein will be required to determine whether it bears homology to other known leukocyte CD antigens. Preliminary studies employing mAb 1B10 as an activating or blocking agent in vitro have failed to reveal an effect on T cell-mediated cytotoxicity or allogeneic recognition, suggesting that the 1B10 molecule is not involved in these classical T cell functions (data not shown). The constitutive expression of 1B10 on tissue resident macrophages and epithelial cells (Table 1) suggests it may function as an adhesion molecules. 1B10 expression, irrespective of it's function, may provide an additional useful marker to detect and to define activated lymphocytes in vivo since 1B10 is maximally expressed at a relatively late stage after activation, after induction of IL-2R, and it's expression is usually maintained on
•
Figure 6. SDS-PAGE analysis of molecules precipitated by mAb IBIO from radio iodinated lysates ofT cells and U937 cells. A. T cell (clone Cn) lysates were precipitated with mAb OKT3 (lanes 1 and 3) or mAb IBIO (lanes 2 and 4) before (lanes 1 and 2) and after (lanes 3 and 4) digestion with N glycosidase F. B. U937 cells precipitated with beads alone (lane 1) or with mAb IBIO, before (lane 2), and after (lane 3) digestion with N glycosidase F respectively. The open arrows indicate the positions of fully glycosylated CD3y (lane IA) and IBIO (lanes 2A and B) molecules precipitated from the lysates, whereas blackened arrows indicate deglycosylated forms of these molecules. The 43 kDa protein co-precipitated by OKT3 (Fig. 6A, lane 1) represents the yo TCR. The nature of the contaminating band migrating at 40 kDa (Fig. 6B, lanes 1-3) is unknown.
60 . 1. BANK et al.
long term previously activated T cells (Fig. 1-3). Furthermore, the levels of 1B10 are independent of VLA-1 and HLA-DR, two classical late activation markers, which may facilitate the detection of VLA-1 low and HLA-DR low previously activated T cell clones in vivo (Fig. 4). Interestingly, in a preliminary study we determined that the level of expression of 1B10 antigen in the PBMC of patients with RA is diminished during therapy with minocycline along with clinical improvement (8). It will be of interest to determine whether the level of expression of 1B10 contributes to T cell migration or localization to the joint space, to the presumed pathogenicity of the cells in the synovium, or to other specific functions of activated T cells in vivo.
References 1. COTNER, T.,]. M. WILLIAMS, L. CHRISTENSON, H. M. SHAPIRO, T. B. STROM, and J. STROMINGER. 1983. Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content.]. Exp. Med. 157: 461. 2. HEMLER, M. E., and]. G. JACOBSON. 1987. Cell matrix adhesion-related proteins VLA-l and VLA-2: regulation of expression on T cells.]. Immunol. 138: 2941. 3. HEMLER, M. E., ]. G. JACOBSON, M. B. BRENNER, D. MANN, and]. L. STROMINGER. 1985. VLA-l: a T cell surface antigen which defines a novel late stage of human T cell activation. Eur. ]. Immunol. 15: 502. 4. LEONARD, W. J.,]. M. DEPPER, T. UCHIYAMA, K. A. SMITH, T. A. WALDMANN, and W. C. GREENE. 1982. A monoclonal antibody appears to recognize the receptor for human T cell growth factor: partial characterization of the receptor. Nature (Lond.) 300: 267. 5. LEDERMAN, S., M. ]. YELLIN, A. M. CLEARY, S. M. FORTUNE, and L. CHESS. 1994. The understanding of contact dependent T-cell helper function in molecular, cellular and physiological detail. Res. Immunol. 145: 215. 6 MASUYAMA, J. 1.,]. S. BERMAN, W. W. CRUISHANK, C. MORIMOTO, and D. CENTER. 1992. Evidence for recent as well as long term activation of T cell migrating through endothelial cell monolayers in vitro.]. Immunol. 148: 1367. 7. IANNONE, E, V. M. CORRIGALL, G. H. KINGSLEY, and G. S. PANAYI. 199 Evidence for the continuous recruitment and activation of T cells into the joints of patients with rheumatoid arthritis. Eur. ]. Immunol. 24: 2706. 8. LANGEVITZ, P., 1. BANK, D. ZENNER, M. BOOK, and M. PRAS. 1992. Treatment of resistant rheumatoid arthritis with minocycline: an open study.]. Rheumatol. 19: 1502. 9. BANK, 1., and L. CHESS. 1985. Perturbation of the T4 molecule transmits a negative signal to T cells.]. Exp. Med. 162: 1294. T cells from a 10. BANK 1., A. TANAY, A. MIGDAL, M. BOOK, and A. LIVNEH. 1995. V"fJ-V02+ patient with Felty syndrome that exhibit aberrant responses to triggering of the CD3 molecule can regulate immunoglobulin secretion by B cells. Clin. Immunol. and Immunopathol. 74: 162. 11. BANK, 1., R. A. DEPINHO, M. B. BRENNER, ]. CASSIMERIS, E ALT, and L. CHESS. 1986. A novel heterodimer on the surface of immature thymocytes. Nature 322: 179. 12. BANK, 1., M. HEMLER, M. B. BRENNER, D. COHEN, V. LEVY,]. BELKO, C. CROUSE, and L. CHESS. 1989. A novel monoclonal antibody, IB3.1, binds to a new epitope of the VLA-l molecule. Cell Immunol. 122: 416. 13. KRICHEVSKY, A., E. G. ARMSTRONG, ]. SCHLATTERER, S. BIRKEN, ]. O'CONNOR, K. BIKEL, S., SILVERBERG,]. W. LUSTBADER, and R. E. CANFIELD. 1988. Preparation and characterization of antibodies to the urinary fragment of human chorionic gonadotrophin ~o subunit. Endocrinology 123: 584.
yo
26 kDa T cell activation antigen . 61 14. KISHIMOTO, T, H. KIKUTANI, A. E. G. VON OEM BORNE, et al. 1997. Leukocyte typing VI. Garland Publishing Inc. New York & London, pp. 114. 15. BANK, I., D. ROTH, M. BOOK, R. BLOCK, P. LANGEVITZ, M. EHRENFELD, and M. PRAS. 1991. Expression and functions of very late antigen 1 in inflammatory joint diseases. J. Clin. Immunol. 11: 29. 16. BANK, I., M. BOOK, and R. WARE. 1994. Functional role of VLA-1 (CD49a) in adhesion, cation dependent spreading, and activation of cultured human T lymphocytes. Cell Immunol. 156: 424. 17. BANK, 1.., M. BOOK, M. HUSZAR, Y. BARAM, I. SCHNIRER, and H. BRENNER. 1993. V'62' T lymphocytes are cytotoxic to the MCF7 breast carcinoma cell line and can be detected among the y'6 T cells that infiltrate breast carcinoma. Clin. Immunol. and Immunopathol. 67: 17. 18. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage n. Nature (Lond.) 227: 680. 19. OKADA, A., I. BANK, L. ROGOZINSKY, Y. TAKIHARA, T M. MAK, L. CHESS, and E W. ALT. 1988. Structure of the y'6 T cell receptor of a human thymocyte clone. J. Exp. Med. 168: 1481. 20. CEBRIAN, M., E. YAGUE, M, RINCON, M. LOPEZ BOTET, M. O. DE LANDAZURI, and E J. SANCHEZ MADRID. 1988. Triggering of T cell proliferation through AIM, an activation inducer molecule expressed on activated human lymphocytes. J. Exp. Med. 168: 162. 21. JENNINGS, L. K., J. T CROSSNO, and M. M. WHITE. 1995. CD9 cluster workshop report: cell surface binding and functional analysis. Leuocyte typing V. White cell differentiation antigens. Editors, SCHLOSMAN, S. E, et al. Oxford University Press, pp. 1249. 22. KROLL, M. H., M. E. MENDELSOHN, J. L. MILLER BALLEN, K. K. J. K. HRBOLlCH, and A. I. SCHAFER. 1992. Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIa and extracellular calcium. Biochem. Biophys. Acta 1137: 248. Dr. I. BANK, Dept. of Medicine F, Chaim Sheba Medical Center, Tel Hashomer 52621 Israel. Fax.: ++972 353 024 90; Tel.: ++972 354 099 69.