antigens following stimulation. A number of surface molecules such as CD69^-^ and 4F2 appear within 4 h after exposure to the activating stimulus, while others,.
Immunology and Cell Biology (1994) 72, 13-21
Characterization of a novel leucocyte activation antigen recognized by the antibody CMRF-37 A. DAISH, B. D. HOCK and D. N. J. HART Haematology/Immunology
Research Group, Christchurch Hospital. Christchurch, New Zealand
Summary We describe a novel activation antigen recognized by the monoclonal antibody CMRF-37. which is absent or only weakly expressed on resting cells but is rapidly induced on T cells, B cells and monocytes following stimuiation. Its kinetics of expression (12-24 h after the addition of the inductive signal) indicate that it is an early activation antigen. The cellular distribution and expression kinetics of the CMRF-37 antigen differ from other known activation antigens and as such should be a useful marker of human leucocyte activation. Key words: activation antigen, monoclonal antibody. Introduetion Resting leucocytes can be activated by a number of different stimuli including antigen, mitogens, cytokines and a number of cell surface directed antibodies. The activation of leucocytes leads to a number of sequential changes in their cell surface components inciuding the induction of molecules either absent or only weakly expressed on resting cells.'"^ Many of these activation antigens such as HLA-DR,' 4F2~ and CD25, the IL-2 receptor alpha^" as well as CD71, the transferrin receptor,'' are not unique to a particular cell lineage but are expressed on a range of lymphoid cell types following activation. There is considerable variation in the induction kinetics of different activation antigens following stimulation. A number of surface molecules such as CD69^-^ and 4F2 appear within 4 h after exposure to the activating stimulus, while others, such as the IL-2 and transferrin receptors, appear after 12 h. The HLA-DR, VLA-1 and VLA-2« antigens appear later still following lymphocyte activation. Given the diverse functional repertoire of leucocytes it is probable that activated cells bear several additional membrane molecules, which are not expressed on resting cells. In this report, we describe a novel monoclonal antibody (mAb). CMRF-37, that defines an early leucocyte activation antigen, absent or only weakly expressed on resting cells. This antigen appears within 12-24 h of stimulation and exhibits a cellular distribution and expression kinetics distinct from Correspondence: Dr D. N. J. Hart, Haematology Department, Christchurch Hospital, Private Bag 4710, Christchurch, New Zealand. Received 11 February 1993; accepted 15 April 1993.
Other previously described early activation antigens, such as the IL-2 and transferrin receptors. Materials and methods The hybridoma producing the CMRF-37 mAb was obtained by fusion between P3X63 mouse myeloma cells and spleen cells from a mouse immunized by an i.p. injection of 2 x 10^ L428 cells (a Hodgkin's disease [HD] derived cell line^) and by a further IV boost 5 weeks later. Following cloning by limiting dilution, the cells were used to prepare ascites fluid in BALB/c mice primed with pristane. The antibody was identified as being of the IgGl subclass K type using the Amersham (Amersham, Buckinghamshire, UK) test kit. Other monoclonal antibodies In addition to the mAb CMRF-37 described in this study, the following mAb were used: 16.4.4 (IgGl, anti-rat class I MHC). CMRF-3 (IgGl, antimycoplasma) and CMRF-1 (IgG2b, p2 microglobulin) were used as controls. Phycoerythrin conjugated Leu 16 (CD20), Leu 1 (CD5) and Leu 45RO (CD45RO) antibodies were obtained from Becton Dickinson, San Jose, CA, USA. The phycoerythrin conjugated CD4 and CD8 were obtained from Dakopatts. Glostrup, Denmark. The CD3 (OKT3), CD4 (OKT4), CD2 (OKTl 1), CD8 (OKT8), L243 (anti-HLA-DR). CD25 (7G7, anti-IL-2 receptor a [IL-2R]) and the 4F2 antibodies were produced from hybridomas obtained from the American Type Culture Collection (ATCC; Rock-
14
A. Daish et al.
ville, MD, USA) and grown in this laboratory. The mAb. FMC63 (CDl9), was kindly provided by Dr Heddy Zola, Flinders Medical Centre, Adelaide, SA, Australia. The CD69 mAb (MLR3) was obtained via participation in the 4th International Leukocyte Differentiation Antigen Workshop. The CD45RA mAb, CMRF11,'*^CD71 mAb, CMRF-2(anti-transfernn receptor) and the CD 14 mAb. CMRF-31, were produced and characterized in this laboratory.
(FITC-SAM; Selenius Laboratories, Melbourne, Vie, Australia) and washed prior to analysis on an EPICS 2 fluorescence cell analyser (Coulter Electronics, Hileah, FL, USA). Double labelling staining involved labelling with mAb and FITC-SAM followed by incubation in mouse serum, prior to adding a phycoerythrin conjugated mAb.
Preparation ofcells
Patching or capping ofthe CMRF-37 Ag was examined by incubating excess purified CMRF-37 mAb with either K562 or L428 cells for 30 min at 4°C. Following washing, cells were labelled with FITC-SAM then resuspended in 10% FCS/RPMI 1640 media and incubated at 37''C for various time intervals, washed, resuspended in 20 mmol/L NaN3/PBS and observed under a fluorescence microscope.
PBMC were prepared by density gradient centrifugation over Ficoll-Hypaque (F/H). Mononuclear cells were obtained from tonsils obtained with informed consent, at routine tonsillectomy. Tonsil T lymphocytes were purified by SRBC rosetting and the nonSRBC rosetting fraction was used as a source of enriched tonsil B cells. Erythrocytes. granulocytes and platelets were obtained by standard methods, as described previously.'^ Normal bone marrow (BM) was aspirated from consenting normal volunteers into preservative free heparin and a BM mononuclear preparation obtained over F/H. Cultured macrophages were obtained by teflon bag culture of PBMC'' in RPMI 1640 medium supplemented with 2.5% AB serum, recombinant granulocyte macrophage-CSF (GM-CSF; gift of Sandoz Pharma. Auckland, New Zealand), macrophage-CSF (M-CSF) or IFN-y (gift of Boehringer Ingelheim, Germany) or alternatively IFN-a (gift of HofTman-La Roche, Basel, Switzerland) at the concentrations indicated. All cell preparations used were greater than 95% viable by trypan blue exclusion. Cell lines The T cell lines HSB-2. Molt 4, and Jurkat; the EBV transformed B cell lines WT49, Mann. Burkitt's lymphoma lines Raji and Daudi; and the myeloid lines K562, HL60 and U937 were grown in 10% FCS/RPMI 1640. The Hodgkin's cell line L428 was obtained from Dr V. Diehl (Klinik fur Innere Medizine. Cologne, Germany) and the KM-H2 and HDLM-2 (grown in 20% FCS/RPMI) cell lines from Dr H. G. Drexler (German Collection of Micro-organisms and Cell Cultures. Braunschweig. Germany). Indirect immunofluorescence Cells (< 10^ per test in PBS) were incubated with mAb for 30 min on ice, washed and incubated with fluoresceinated F(ab')2 sheep antimouse Ig at 30jig/mL
Mobility ofthe CMRF-37 antigen in the plasma membrane
Stimulation of leucocytes with mitogens and cytokines Optimal conditions for lymphocyte activation as established using [^H]-thymidine uptake assays were PHA (Sigma, St Louis. MO, USA) at 5 ^ig/mL, CD3 mAb (OKT3) at 250 ng/mL plus IL-2 at 100 U/mL, the phorbol ester, PMA (Sigma) at 25 ng/mL plus the calcium ionophore Cal A23187 (Sigma) at 500 ng/mL Cells were cultured in 10% AB serum/RPMI containing the above concentrations of stimuli and at fixed time intervals analysed by flow cytometry. Enzyme studies The enzyme susceptibility of the CMRF-37 antigen was tested by incubating (37=C, 1 h) L428 and U937 cells in 2 mL PBS containing DNAse (1 mg/mL, Sigma) and either TPCK-treated papain (50ng/mL, Sigma), pronase (50|.ig/mL, Calbiochem. San Diego, CA, USA) or neuraminidase (0.1 U/mL, Boehring, Marburg, Germany). Cells were washed ( x 3) in PBS and analysed by flow cytometry. Im m unoprecipita tion Cells were surface labelled with biotin'^^ or biosynthetically labelled with ^^S'"" prior to solubilization with either 0.6% NP40, 1% NP40, 1% Triton X-100 or 0.25% CHAPS. Solubilized proteins were immunoprecipitated by a modification ofthe method of Tedder er ai'^ using rabbit anti-mouse Ig covalently coupled to CNBr activated sepharose 4B (RAM sepharose) Following preclearing ofthe lysate by 3 h incubation with
CMRF-37: A novel activation Ag
15
RAM-sepharose, solubilized proteins were immunoprecipitated by ovemight incubation with RAMsepharose precoated with the appropriate mAb. Bound antigen was eluted with sample buffer and analysed by 7.5-20% gradient SDS-PAGE (reducing) in combination with either autoradiography or westem blotting. Biotin-labelled proteins were visualized after transfer to a nitrocellulose membrane by streptavidinbiotinylated horseradish peroxidase complex'^ and the Enhanced Chemiluminescence Detection System (Amersham) as described by the manufacturers.
with PBMC lymphoid populations. Tonsil lymphocytes were weakly positive with up to 20% of the T lymphocytes staining with CMRF-37 and the percentage ofcells labelled appeared to parallel the expression of other activation antigens (e.g. IL-2R) on the tonsil cells (data not shown). Monocytes from normal individuals showed considerabie variation in labelling; from nil to 100% weak positive. Within bone marrow 6-11 % of the mononuclear cells stained with CMRF-37 (data not shown.
Results
Reactivity of CMRF-3 7 with human cell lines
Reactivity of CMRF-37 with normal haemopoietic cells The CMRF-37 antigen has a very limited distribution on resting haemopoietic cells (Fig. 1). The CMRF-37 mAb did not label erythrocytes. platelets or granulocytes and was either negative or only weakly reactive Control
CMRF 37
RBC
Granulocytes
Monocytes
PBL
Platelets
Tonsils
I »
The reactivity of the mAb, CMRF-37. on cultured human cell lines was tested in parallel with other clustered activation antigens (Table I). CMRF-37 reacted with all the T cell lines tested, with the strongest expression observed on Jurkat cells. Of the B cell lines tested, WT49 and the Burkitt's lymphoma cell lines, Raji and Daudi, all showed weak staining, while the Epstein Barr virus (EBV) transformed B cell line, Mann, was essentially negative. Both the myeloid lines, K562 and U937 reacted with CMRF-37. L428 was the only HD-derived cell line that was clearly positive for CMRF-37. Of the other two HD cell lines tested. KM-H2 was weakly positive and HDLM-2 had a weak shoulder of positive staining (Table 1). The CMRF-37 antigen distribution on cell lines clearly distinguished it from the CD69, CD25 (IL-2 receptor) and CD71 (transferrin receptor) antigens (Table 1). The mAb 4F2 (also generated following immunization with L428) was examined and was readily distinguished from CMRF-37 on the basis of its strong reactivity with the B cell line Mann. Kinetics of CMRF-37 expression on T lymphocytes
Log fluorescence
Fig. 1. Flow cytometry analysis of haemopoietic cell populations labelled with CMRF-37 and the isotype matched negative control antibody 16.4.4. Each cell population was studied at least three times on different individuals and the typical profiles obtained on cells from one donor are shown.
The expression of CMRF-37 antigen on both peripheral blood and tonsil T lymphocytes was examined following activation with PHA. CD3 + IL-2 or PMA + Cal. PBMC were incubated with these stimulatory agents and the T lymphocyte surface phenotype was analysed at various times thereafter, by double labelling with CD5 (red) and CMRF-37 mAb (green). Preliminary experiments comparing CD2, CD3 and CD5 mAb established that CD5 was a satisfactory marker for T cell labelling (and most appropriate for monitoring of PBMC activation) with inconsequential numbers of CD5 positive B cells noted in the preparations. Resting T lymphocytes were CMRF-37 negative. However all three methods of lymphocyte activa-
16
A. Daish et al.
Table I. Activation antigen expression on cell lines. Cell lines Jurkat HSB2 Molt 4 Mann WT49 Raji Daudi K562 U937 L428 KM-H2 HDLM-2
CMRF-37 % (MFI) 99 99 98 2 81 99 98 93 93
99 30 11
(343.4) (65.52) (109.7) (3.467) (15.64) (42.53) (35.85) (82.82) (24.00) (344.7) (4.452) (3.103)
%
CD71 (MFI)
89 96 99 96 95 99 93 90 46 79 100 90
(28.52) (83.74) (93.75) (51.77) (57.21) (53.27) (20.57) (91.96) (11.64) (28.77) (247.0) (54.45)
% 1
2 1 1 8
5 1 0 34 2 19 99
CD25 (MFI) (1.936) (2.413) (1.833) (2.304) (3.304) (2.811) (4.752) (4.756) (9.179) (3.402) (3.686) (7.714)
% 13 91
50 12 12 2 1 18 97
23 78 4
CD69 (MFI) (4.545) (34.91) (8.256) (3.545) (3.545) (2.831) (5.156) (13.31) (45.42) (8.489) (11.15) (4.727)
Results are expressed as per cent cells positive and figures in parentheses are mean fluorescent intensities (MFI) on standardized fluorescence activated cell sorter settings.
tion induced expression of the CMRF-37 Ag within 48 h, on both tonsil and blood T lymphocytes (Fig. 2). Peak expression of the CMRF-37 Ag was seen on PBMC T lymphocytes stimulated with PHA at 48 h (Fig. 2a) with expression returning to baseline at 96 h. Stimulation with CD3 + IL-2 induced expression more slowly and the CMRF-37 Ag levels continued to increase steadily up to 144 h (Fig. 2c). In contrast, PMA + Cal caused a rapid increase in expression ( 95% CD 19 and CD20 positive) was studied using PMA + Cal as the inductive stimuli. PMA + Cal activation of tonsil B lymphocytes induced a slow increase in CMRF-37 expression which was maximal at days 3 and 4 (60-80% of cells positive) and returned to baseline levels by day 7 (Fig. 4). This was in contrast to IL-2R expression, which increased slowly over the 7 day time course with the intensity of staining (i.e. surface antigen density) continuously increasing. Expression of CMRF-37 antigen during monocyte to macrophage differentiation and activation CMRF-37 Ag expression was monitored during monocyte to macrophage differentiation, induced by culture of monocytes in teflon bags" containing either AB serum (Fig. 5), GM-CSF, M-CSF, IFN-y or IFN-a.
17
CMRF-37: A novel activation Ag
PHA
IOOT
CD3 + IL2
100
(d)
CD3 + IL2
80-
60-
40
20
24
lOOi
(0
48
72
96
120 144
168
PMA + CA
80-
60
40
11
< 20 n
t
24
t~
t"
48
72
4 —\
95
1
120 144
1
1
168
Time (h) Fig. 2. The kinetics of CMRF-37 Ag expression on peripheral blood T lymphocytes and tonsil T lymphocytes. The percentage of CMRF-37 positive T (CD5 positive) lymphocytes (mean ± s.e.m.) were measured by double labelling at the indicated times afYer activation with various stimuli, (a) blood. PHA, n = 3: (b) tonsil. PHA, n = 3; (c) blood, CD3 + IL-2, « =• 3; (d) tonsil, CD3 + IL-2, « = 3; (e) blood, PMA + Cal. n = 3; (f) tonsil, PMA + Cal, n = 3). CMRF-37 expression on activated cells (O), CMRF-37 on cells that are not stimulated but cultured in media alone (A).
Flow cytometry analysis showed that the percentage of CMRF-37 positive cells increased to 100% (mean fluorescence intensity; MFI = 40) within the first 24 h of culture, in each case with the apparent surface antigen density intensity increasing through to 14 days (MFI= 130). This increase was again independent of the cytokine stimulus used. The MFI of surface p2 microglobulin staining which can be used as a crude measure of cell volume increased at the same rate as CMRF-37 after 24 h, suggesting that the apparent in-
crease in CMRF-37 intensity, as monocytes differentiate into macrophages. may be due to an increase in cell size rather than increased antigen density.
Patching ofthe CMRF-37 antigen The CMRF-37 labelled Ag was observed to patch on the L428 and K562 cell lines within 30 min and cap within 60 min. Following ovemight culture at 37°C
A. Daish et al.
18
CD3 + IL2
PHA
100-1 80-
0
48
72
96
120 144-
0
24
PMA + CAI
CA
o o.
24
72
4fl
96
120
PMA + CAI
1001
100-,
80-
60-
tn CC
S o
96
120
4
144
6
8
10 12 14 16 18
Time (h) Fig. 3. The kinetics of the CMRF-37 Ag compared to that of other established activation antigens on peripheral blood T iymphocytes following stimulation. The percentage of CMRF-37 positive T (CD5 positive) T cells was measured by double labelling at different times after activation. The time courses were repeated three times and one representative experiment is shown, (a) PHA; (b) CD3 + IL-2; (c) PMA + Cal. In (d) a short time course is shown following stimulation with PMA + Cal. Activation antigens labelled: CD69 (•), CMRF-37 Ag (O), HLA-DR (A). IL-2R (D) and CD7I (•).
complete internalization of the antigen was observed on the majority ofcells. Characterization ofthe CMRF-37 antigen Enzyme digestion studies on the cell lines L428 and U937 showed that the CMRF-37 antigen was pronase and papain sensitive (as monitored by flow cytometry) and that binding was not affected by neuraminidase treatment (data not shown). In an attempt to define the CMRF-37 antigen further, ^^S and biotin labeiied iysates of the L428 and Raji ceii lines, as well as PMA + Cal activated PBMC were immunoprecipitated with the CMRF-37 mAb. Additional cell lysates were used to immunoprecipitate several other characterized activation antigens. We were unable to immunoprecipitate the CMRF-37 Ag despite using a range of solubilization conditions and successfully immunoprecipitating the HLA-DR,
CD69, IL-2R and 4F2 antigens (Fig. 6). These results further support our view that the CMRF-37 mAb recognizes a new leucocyte activation antigen. However, in the absence of definitive biochemical data, it remains possible that the CMRF-37 mAb reacts with an undescribed epitope of a known activation antigen to produce the novel cellular reactivity described above.
Ability of CMRF-37 antigen to inhibit in vitro cellular function To determine whether CMRF-37 can abrogate leucocyte function, a number of antibody blocking experiments were carried out. When added to cultures of monocytes, the CMRF-37 antibody had no detectable effect on either plastic adherence or phagocytosis of latex particles {n = 2). Similarly, the presence of CMRF-37 mAb in mixed leucocyte reactions (MLR)
CMRF-37: A novel activation Ag
CMRF 37
CONTROL 1 468 DAYO
i
) 235
CD25 1 629
I
19
3-055
L
1 2
1 440
3-224
3
4
5
11697-
DAY 1
66 -
Ik. 1-284
8-853
40 59
DAY 4
1
45-
ill. 4 578
f*M H
45 5B
DAY 7
A
M Log Fluorescence
Fig. 4. Flow cytometry analysis of tonsil B lymphocyte (> 95% CD19. CD20 positive) activation, following stimulation with PMA + Cal. Profiles obtained after labelling with control. CMRF-37 and the anti-IL-2R (CD25) mAb are shown. The MFI is indicated at the top right hand comer of each histogram. Three time course experiments were performed and one representative experiment is shown.
29-
Fig. 6. Comparative immunoprecipitation analysis of CMRF-37 and other activation antigens. Biotin labelled PMA + Cal activated PBMC were lysed with 0.6% NP40 and precipitated with the mAb CMRF-37 {lane 1). 4F2 (lane 2). anti-CD69 (lane 3), anti-HLA-DR (lane 4) and anti-CD25 (lane 5). The precipitates were analysed by immunoblotting in combination with a chemiluminescent detection system. Moiecuiar mass standards (kDa) are indicated on the left. did not alter the allogeneic T lymphocyte proliferative
lOOOn
response (n = 3). Discussion
100-
o
1 10-
DO
DI
D7
D14
Fig. 5. MFI of CMRF-37 labelling during monocyte to macrophage differentiation induced by culture in teflon bags containing 2.5% AB serum. Note that the MFI is plotted as a log scale. Bars indicate s.e.m. n = 4. (M) Control; (•) CMRF-37; (D)CD14.
Activation of human leucocytes is associated with the expression of new surface antigens. In this report, we describe a novel activation antigen recognized by the mAb CMRF-37. This cell membrane antigen is absent or only weakly expressed on peripheral blood and tonsil lymphocytes but is rapidly upregulated following activation. A number of other activation antigens with broad leucocyte reactivity have been described.'"^ but only a few activation antigens, notably 4F2.-^ CD69.**-^ IL-2R,-'''^ the transferrin receptor*^ and the MEI4/D12 antigen" have a similarly limited expression on resting lymphocytes. The possibility ihat CMRF-37 reacts with a known activation antigen was tested by comparing the cellular reactivity and kinetics of expression of CMRF-37 with a number of well-defined activation molecules. The CMRF-37 Ag is detected 12-24h after the onset of
20
A. Daish et al.
stimulating peripheral blood T lymphocytes with PHA, PMA + Cal or CD3 + IL-2. The expression kinetics and percentage staining of CMRF-37 during PHA and CD3 + IL-2 T lymphocyte stimulation is quite distinct from that of CD69 and IL-2R and resemble those of HLA-DR and the transferrin receptor most closely. PMA + Cal activation of T cells, however, showed clear differences in the induction of CMRF-37 as compared to HLA-DR. transferrin receptor and CD69 antigens. A considerably higher percentage of PHA activated T lymphocytes express the IL-2R as opposed to the CMRF-37 antigen, and this further distinguishes the CMRF-37 antigen from the ME14/D12 and 4F2 activation antigens, which have been reported as being expressed on a similar percentage of cells as IL-2R following PHA activation.'"^ These activation studies have again documented differences between tonsil T lymphocytes and peripheral blood T lymphocytes in their expression of activation antigens in response to the same stimuli. These differences in the levels of antigen expression following activation, for example, the CMRF-35 Ag.'^ may reflect the fact that tonsil T lymphocytes belong to the extravascular tissue compartment or have been exposed to previous activation via the pathological processes necessitating tonsil extraction. Expression ofthe CMRF-37 Ag can also be induced on monocytes and B cells with activation. Therefore it is not surprising, in view of its known normal cell distribution and documented activation potential, that the CMRF-37 antigen was broadly distributed on human cell lines, being detected on cell lines of both T, B and myeloid lineage. Again the spectrum of CMRF-37 reactivity on human cell lines clearly distinguishes it from the CD69. CD71, CD25 and 4F2 mAb. The ME14/D12 antigen, which has broadly similar expression kinetics and cellular distribution to that of CMRF-37, can also be distinguished from the CMRF-37 antigen on the basis of its reported lack of expression on the strongly CMRF-37 expressing cell lines Jurkat and Molt 4.'^ The results of these studies on the cellular distribution and expression kinetics of the CMRF-37 Ag strongly suggest that the CMRF-37 mAb identifies a novel activation antigen. Enzyme digestion studies indicated that the CMRF-37 antigenic epitope is protein associated; however, we failed to immunoprecipitate the CMRF-37 antigen, despite our ability to precipitate other previously characterized activation antigens using a range of labelling and detergent solubilization systems. This provides additional supportive evidence that the CMRF-37 antigenic determinant is distinct from those of other characterized activation antigens.
Nonetheless, it must be conceded that until definitive molecular information as to the nature of antigen is obtained, this question will remain in doubt. Attempts to isolate a cDNA clone encoding the molecule using expression cloning techniques are underway.'^ Isolation of such a clone will allow further antibodies to be developed, as well as functional studies, such as adhesion, to be carried out. The inability of the mAb CMRF-37 to block a MLR does not imply that this antigen is unimportant in cell activation, as the failure to inhibit may be related to failure to bind to functionally related epitopes, low mAb affinity compared to natural ligands or inadequacies in the assay system. In view of its strong upregulation following cellular activation, it would be most unlikely that the CMRF-37 Ag was not involved in some aspect of leucocyte function. The challenge is to find that function. In that regard it is notable that most antibodies to the transferrin receptor do not block binding of transferrin or inhibit cell proliferation.'^ In summary, the mAb CMRF-37 defines a previously uncharacterized early activation antigen absent (or only weakly expressed) on resting cells, but which is induced rapidly on T lymphocytes, B lymphocytes and monocytes following activation. Although the function of this antigen is as yet unknown, its unique cellular distribution and expression kinetics make it a useful tool in the study of leucocyte activation. Acknowledgements The authors wish to thank their colleagues who assisted with this work and the volunteers who provided blood samples. This work was supported by the McClelland Trust, the Lochmaben Trust and the Health Research Council of New Zealand. References 1. Ko. H.. Fu, S. M., Winchester, R. J.. Yu. D. T. Y. and Kunkel. H. G. 1979. Ia determinants on stimulated human T lymphocytes; Occurrence on mitogen-andantigcn-activated T cells. / E.xp. Med. 150: 246-255. 2. Haynes, B. F., Hemler. M. E., Mann, D. L. et ai 1981. Characterisation of a monoclonal antibody (4F2) that binds to human monocytes and to a subset of activated lymphocytes. ///??m(m()/. 126: 1409-1414. 3. Uchiyama, T., Border. S. and Waldman. T. A. 1981. A monoclonal antibody (Anti-Tac) reactive with activated and functionally mature human T cells. ./. Immunoi 126: 1393-1397. 4. Muraguchi, A., Kehrl, J. H., Longo, D. L., Volkman, D. J., Smith, K. A. and Fauci. A. S. 1985. Interleukin 2 receptors on human B cells. Implications for the role of
CMRF-37: A novel activation Ag
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the CDI5 mAb CMRF-7 and 27. Pathology 20: 24-31. Andreesen, R., Brugger, W., Scheibenbogen, C et ai 1990. Surface phenotype analysis of human monocyte macrophage maturation. / Leucocyte Biol. 47: 490-497. Cole, S. R., Ashman, L. K. and Ey, P. L. 1987. Biotinylation: An alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates. Mol. Immunol. 24: 699-705. Tedder, T. F., Mclntyre, G. and Schlossman, S. F. 1988. Heterogeneity in the Bl (CD20) cell surface molecule expressed by human B-lymphocytes. Moi Immunoi 25: 1321-1330. Carrel, S., Salvi, S., Isler, P. et al. 1990. gp33-38, An early human T cell activation antigen. / Immunoi 144: 2053-2062. Daish, A., Starting, G. C , McKenzie, J, L., Nimmo. J. C , Jackson, D. G. and Hart, D. N. J. 1993. Expression of the CMRF-35 antigen, a new member ofthe immunoglobulin gene superfamily, is differentially regulated on leucocytes. / Immunoi 79: 55-63. Seed, B. and Aruffo. A. 1987. Molecular cloning of the CD2 antigen, the T-celi erythrocyte receptor, by a rapid immunoselection procedure, Proc. Natl Acad. Sci. USA 84: 3365-3369. Mendelsohn. J.. Trowbridge. I. and Castagnda. J. 1983. Inhibition of human lymphocyte proliferation by monoclonal antibody to transferrin receptor. Blood 62: 8 2 1 826.
