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Identification of a human T lymphocyte surface protein associated with the E-rosette receptor

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Abstract

We describe a new monoclonal murine antibody that reacts with a 50,000-mol wt polypeptide that appears to be present on all E-rosetting cells. We conclude that this antigen is either identical to or closely associated with the E receptor because of (a) the high degree of concordance between E-rosette formation and 9.6 antigen expression, (b) the inhibition of rosette formation by preincubation of cells with 9.6 antibody, and (c) the observed failure of cells lysostripped of 9.6 antigen to form E-rosettes. This last finding suggests cocapping of 9.6 antigen and the E receptor.
Brief Definitive Report
IDENTIFICATION OF A HUMAN T LYMPHOCYTE SURFACE
PROTEIN ASSOCIATED WITH THE E-ROSETTE RECEPTOR
BY MALEK KAMOUN, PAUL J. MARTIN,* JOHN A. HANSEN,
MELISSA A. BROWN, ANTHONY W. SIADAK, ANO ROBERT C. NOWINSKI
From the Fred Hutchinson Cancer Research Center; and the Puget Sound Blood Center, Seattle, Washington
98104; and the Department of Medicine, Division of Oncology, University of Washington School of
Medicine, Seattle, Washington 98195
Human thymus-dependent lymphocytes (T cells) are defined by their ability to
form spontaneous rosettes (E-rosettes) with sheep erythrocytes (SRBC) (1-3). Re-
cently, several murine monoclonal antibodies that recognize distinct differentiation
antigens on human T cells have been described (4-11). We report here a monoclonal
antibody, designated 9.6, that identifies a 50,000-dalton surface protein that appears
to be present on all E-rosette-forming cells. Blocking and lysostripping experiments
indicate that this monoclonal antibody reacts either with the E receptor itself or with
a closely associated structure.
Materials and Methods
Cells.
Mononuclear cells from peripheral blood (PBL) or bone marrow were obtained from
normal volunteers and isolated by centrifugation over Ficoll-Hypaque (LSM; Litton Bionetics
Inc., Kensington, Md.). T cells were enumerated by rosetting with 2-aminoethylisothiouronium
bromide hydrobromide (AET)-treated SRBC (SRBC^v..r) in medium (RPMI-1640) with 12%
fetal calf serum (12). For the isolation of purified T cells, PBL were passed over nylon wool (11)
and then incubated with SRBCA~--v. Rosette-forming T cells (E +) were separated from non-T
cells (E-) by centrifugation over Ficoll-Hypaque. T cells were recovered from the rosettes by
lysis of SRBC in Tris-buffered 0.83% ammonium chloride. Normal human thymocytes were
prepared from thymus specimens obtained in the course of corrective open heart surgery from
children < 14 yr old. Long-term-cultured T cells, stimulated by alloantigen, were maintained in
medium supplemented with T cell growth factor (TCGF) (13).
A selected panel of cultured lymphoid cell lines was used for serological analysis. Included
were leukemic T cell lines (CEM, HSB2, 8402, Jurkat, Molt-4F, and KE37), the leukemic pre-
B cell line NALM-6, Epstein-Barr virus-transformed B-lymphoid cell lines (PA-3, SB, 8392,
HA, Swei), and the Burkitt's lymphoma ceil line Daudi.
Leukemic blasts from peripheral blood of patients with acute lymphocytic leukemia (ALL),
were separated over Ficoll-Hypaque, and cryopreserved. Cells were obtained either at presen-
tation or relapse when the leukocyte count was >20,000/mm s and >90% of the cells were blasts.
Subdivision of ALL into T and null cell types was based on the clinical criterion of a thymic
mass, and on determination of whether the leukemic blasts formed E-rosettes or expressed ia-
like antigen.
Immunizations, Fusion, and Screening.
The clone of hybrid cells producing the antibody
described here was isolated from a previously described fusion experiment (9). Briefly, spleen
cells from BALB/c mice immunized with human peripheral blood lymphocytes were fused
with BALB/c MOPC21 NSI/1 myeloma cells. Antibody production by hybrid cells was assayed
by testing tissue culture supernates in a complement-dependent microcytotoxicity assay against
normal peripheral blood T cells, a B-lymphoid cell line, and continuously cultured T cells, all
obtained from the same donor. One culture fluid was cytotoxic for both the normal and
cultured T cells but nonreactive with the autologous B-lymphoid cell line. Cells from this
* Junior Faculty Clinical Fellow of the American Cancer Society.
J. Exp. MED. © The Rockefeller University Press • 0022-1007/81/01/0207/06 $1.00 207
Volume 153 January 1981 207-212
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208 KAMOUN ET AL. BRIEF DEFINITIVE REPORT
culture, designated 9.6, were serially cloned four times by limiting-dilution and then inoculated
intraperitoneally into Pristane-primed (Aldrich Chemical Co., Inc., Milwaukee, Wis.)
BALB/c mice for the production of antibody-containing ascites fluid. The immunoglobulin
produced by the 9.6 hybrid was identified as IgG2b by immunodiffusion.
Serological Analysis. Methods for complement-dependent microcytotoxicity, quantitative
cytotoxicity (using trypan blue as indicator), and indirect immunofluorescence assays have
been previously described (9, 11). A fluorescein-conjugated affinity-purified goat anti-mouse
IgG serum (Litton Bionetics Inc.) was used for indirect immunofluorescence. In some experi~
ments rhodamine-conjugated goat F(ab')2 fragments specific for mouse IgG were used (this
reagent was generously provided by Dr. Shu Man Fu, The Rockefeller University, New York).
Comparative studies were carried out using three additional monoclonal antibodies recognizing
distinct markers for human lymphocytes. Antibody 9.3 reacts with a 45,000-dahon surface
protein expressed by 70-80% of peripheral E + cells (9); antibody 10.2 reacts with a 65,000- to
67,000-dahon surface protein expressed by 85-95% of peripheral blood E + cells (11); and
antibody 7.2 reacts with a framework determinant of the human Ia bimolecular complex (9).
Blocking of E Rosette Formation and Lysostrippmg. Cells were incubated in medium that
contained monoclonal antibody for 30 min at 22°C and then washed three times. In blocking
experiments, these cells were subsequently assayed for E rosette formation. In lysostripping
experiments, treated cells were incubated with affinity-purified goat anti-mouse IgG serum
(fluorescein conjugated) for 45 rain at 37~'C, washed three times, and then tested for rosette
formation.
Immune Prectpitation of Cell Membranes. Cells of the leukemic T cell line Jurkat were washed
twice in phosphate-buffered safine, pH 7.2, and surface labeled by the 125I-lactoperoxidase
method (14) with minor modification (15). Labeled ceils were disrupted in cell lysis buffer that
contained 0.5% Nonidet P-40 (15) and 2% of the protease inhibitor aprotinin (Sigma Chemical
Co., St. Louis, Mo.). The lysate was cleared of nonsolubilized cellular structures and free 125I by
uhracentrifugation and chromatography on a Sephadex G-25 column as described elsewhere
(16).
Before use in immune precipitation analysis, the cell lysate was treated by sequential
incubation (1 h each at 4°C) with AKR normal mouse serum (1:40 dilution) and Staphylococcus
aureus, Cowan I strain (30 mg) (17). The S. aureus was removed from the reaction mixture by
centrifugation at 1,500 g for 10 min. The concentration of sodium dodecyl sulfate (SDS) in the
lysate was adjusted to 0.1% and the immune precip!tation reactions initiated by the addition
6
of 5 #1 of appropriately diluted antibody to 95-#1 (I0 cpm) aliquots of radiolabeled lysate. The
mixture was incubated for 1 h on ice and the reaction then terminated by fhe addition of 3.5
mg S. aureus for 30 min on ice. The S. aureus pellet was washed five times in buffer that contained
0.5% NP-40 and 0.1% SDS (15). Radiolabeled proteins bound to the pellet were extracted by
incubation with 50 #1 of sample electrophoresis buffer (0.062 M Tris-HCl, 2% SDS, 10%
glycerol, 5% 2-mercaptoethanol, and 0.02% bromphenol blue, pH 6.8) for 5 min at 100°C and
then analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS (SDS-
PAGE) in 10% slab gels (18). Radiolabeled bands in the dried gel were identified by
radioautography (Kodak NS-2T film [Eastman Kodak Co., Rochester, N. Y.] with GAFMED
Rarex B Mid Speed intensifying screen [GAF Corp., New York] with exposure at -70°C) (19).
Results and Discussion
The 9.6 antigen was present on peripheral blood T cells and thymocytes, but was
not detected on mononuclear cells of peripheral blood or bone marrow after removal
ofT cells by E-rosetting (Table I). T cells purified by passage over nylon wool followed
by centrifugation of E + cells over Ficoll-Hypaque were >99% positive with 9.6
antibody. Thymocytes were also >99% positive for the 9.6 antigen. Cells from B
lymphoid lines were uniformly negative.
A high degree of concordance between E-rosette formation and expression of 9.6
antigen was observed in testing cells from a variety of sources (Table I). Similar
numbers of 9.6-positive and E + cells were found in PBL, thymus, bone marrow, and
long-term-cultured T cells. Expression of 9.6 antigen also correlated with E-rosetting
in cells of leukemic T cell lines. Only the E + leukemic T cell lines 8402, Jurkat, and
Molt-4F were 9.6 positive, each demonstrating equal numbers of E + and 9.6-positive
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KAMOUN ET AL. BRIEF DEFINITIVE REPORT 209
TABLE I
Comparison of the Number of E-Rosetting and Monoclonal Antibody 9.6-Positive Cells
in Normal and Cultured Lymphoid Cells*
Percent E ÷ Percent 9.6 positive
Peripheral blood lymphocytes 72
Purified T cells 99
Non-T cells <1
Thymocytes >99
Bone marrow cells 9 17
T cell depleted < 1
B-lymphoid cell lines$§ <1
Cultured T cellsll >99
Leukemic T cell lines§
GEM < 1
8402 23
Jurkat 90
HSB-2 < 1
KE37 <1
Moh-4F 17
Mofi-4F, E ÷ enriched 85
Moh-4F, E ÷ depleted 3
(9) 68 (9)
(5) 99 (5)
(3) <1 (3)
(3) >99 (3)
(3) 10 15 (4)
(2) <1 (2)
(7) <l (7)
(3) >99 (:3)
<1
20
98
<1
<1
20
93
4
* Assays for E-rosette forming cells E ÷ were performed with SRBC^m-. Testing with 9.6 antibody
was performed by indirect immunofluorescence. The number of donors tested is indicated in
parentheses. Where appropriate, results are expressed as the mean (or range) of multiple samples.
:~ NALM-6, Daudi, PA3, SB, 8392, HA and Swei.
§
Cell lines CEM and PA3 were obtained from Dr. D. Mann, National Institutes of Health,
Betbesda, Md.; HSB-2, SB, and Jurkat from Dr. W. D. Peterson, Wayne State University,
Detroit, Mich.; 8402, 8392, Molt-4F, and NALM-6 from Dr. J. Minowada, Roswell Park
Memorial Institute, Buffalo, N. Y.; KE37 from Dr. S. M. Fu; and Daudi from Dr. W. Newman,
Fred Hutchinson Cancer Research Center, Seattle, Wash. Derivation of B-lymphoid cell lines
HA and Swei has been previously described (20).
II Normal T cells stimulated with alloantigen and cultured >3 mo in TCG Factor.
TABLE II
Effect of Antibody 9.6 on E-Rosette Formation
Cells treated
E-Rosette formation after incubation with
antibody*
Control Antibody 10.2 Antibody 9.6
Peripheral T cells:~ 86 92 < 1
Thymocytes 99 98 < 1
Cultured T cells 99 99 < 1
Leukemic T cell line, Jurkat 92 96 < 1
PHA blasts§ ND 94 < I
* Results expressed as the percent rosette-forming cells. Ascites fluids that contained' antibody
10.2 or 9.6 were tested at a dilution of 1 : 1,000. Rosette inhibition with antibody 9.6 persisted to
a dilution of 1:64,000. ND, not determined.
:~ Nylon wool-nonadherent peripheral blood lymphocytes.
§ Nylon wool-nonadherent peripheral blood lymphocytes were cultured for 72 h in complete
medium with 10% pooled human serum and 12 #g/ml phytohemagglutinin (PHA-P: Burrnughs
Wellcome & Co., Research Triangle Park, N. C.).
cells. Molt-4F cells, enriched for E + cells were 9.6 positive, whereas Molt-4F cells
depleted of E + cells were 9.6 negative. Cells from patients with T cell ALL whose
leukemic blasts formed E-rosettes were 9.6 positive. Leukemic blasts from patients
with null cell ALL were 9.6 negative.
The association between the E receptor and the 9.6 antigen was further investigated
by blocking experiments. Cells preincubated with antibody 9.6 did not form E-rosettes
(Table II). Incubation with either of the two other monoelonal T cell antibodies, 9.3
(data not shown) or 10.2, did not inhibit E-rosetting. Rosette inhibition by antibody
9.6 paralleled its activity in a complement-mediated cytotoxicity assay.
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210
KAMOUN ET AL. BRIEF DEFINITIVE REPORT
TABLE III
Lysostripping
Treated cells tested by indirect
Lysostripping E + immunofluorescence:~
antibody* 9.6 10.2
None 94 94 91
9.6 4 0 92
1(I.2 95 77 0
* Cells were incubated with monoclonal antibody diluted in medium for 0.5 h at 22°G
and washed three times. Cells were then incubated with fluorescein-conjugated goat
anti-mouse lgG serum for 45 min at 37°C and the washing was repeated. Fluorescein
staining was detected only in polar caps. Rosetting was assayed with SRBG^m,, Numbers
in the Table repre~nt the percentage of rosetting cells.
:~ To determine whether any uncapped mouse immunoglobulin remained on the cells
after lysostripping, cells were stained with rhodamine-conjugated goat F(ab')2 fragments
specific for mouse immunoglobulin. No rhodamine staining could be detected outside
the polar caps. Treated cells were also tested with 9.6 antibody and 10.2 antibody by
indirect immunofluorescence to determine whether the respective antigens had been
completely capped. The rhodamine-conjugated antiserum facilitated distinction be-
tween staining of previously formed polar caps and circumferential staining of antigens
that remained distributed on the cells. Numbers in the Table represent percent positive
cells.
Lysostripping of cells incubated with monoclonal antibody was performed to
determine whether the E receptor and the 9.6 antigen could be distinguished by co-
capping. Cells lysostripped of 9.6 antigen did not form E-rosettes, whereas cells
lysostripped of the 10.2 antigen were still capable of rosette formation (Table III).
Capping of antigen-antibody complexes appeared complete, as assessed by indirect
immunofluorescence (Table III) and quantitative cytotoxicity (data not shown). It
appeared, therefore, that the failure of cells stripped of 9.6 antigen to form E rosettes
was not a result of residual blocking antibody. When cells were lysostripped of 9.6
antigen and then incubated in fresh medium at 37°C for 18 h, they again expressed
9.6 antigen and formed E-rosettes.
Results of precipitation assays with antibody 9.6 and an 12~I-labeled lysate of the
leukemic T cell line Jurkat are presented in Fig. 1. The antibody precipitated a single
polypeptide of -50,000 mol wt. In this same assay are shown the results with
antibodies 9.3 and 10.2, which precipitated proteins of 45,000 and 67,000 daltons,
respectively. The 45,000-dalton protein precipitated by 9.3 antibody was not associ-
ated with flz-microglobulin. This was in contrast to that observed with precipitates
formed by antibodies against HLA heavy chain (W6/32) and fl2-microglobulin.
Control reactions performed with normal mouse serum and monoclonal antibody 7.2
against human Ia antigen failed to show precipitation with the cell lysate. In
additional precipitation assays with 12~I-labeled lysates of normal T cells (data not
shown), the 9.6 antibody demonstrated essentially similar results to those found with
the Jurkat cell line, although the antigen precipitated from normal T cells showed a
slightly more disperse pattern on the gel. These results suggested that the antigen
precipitated by 9.6 may have variable glycosylation patterns on T cells from different
sources. Alternatively, the different pattern on gels might reflect mild proteolytic
degradation of the antigen that varied from one T cell type to another.
Summary
We describe a new monoclonal murine antibody that reacts with a 50,000-mol wt
polypeptide that appears to be present on all E-rosetting cells. We conclude that this
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KAMOUN ET AL. BRIEF DEFINITIVE REPORT 211
FtG. 1. Radioimmune precipitation assays with monoclonal antibodies and a lzSI-surface-labeled
lysate of the leukemic T cell line Jurkat. Aliquots of radiolabeled cell lysate were tested in immune
precipitation assays with: (1) normal mouse serum (1:50 dilution); (2) monoclonal 7.2 antibody
from ascites fluid (1:500) which recognizes a monomorphic determinant of human la antigens (9);
(3) rabbit anti-human/32-microglobulin serum (Dakopatts, Copenhagen, Denmark; 1:50 dilution);
(4) monoclonal antibody W6/32 (Allied Chemical Corp., Morristown, N.J.; 1:100 dilution) which
recognizes a monomorphic determinant of the HLA-A,B,C heavy chain (21); (5) monoclonal 9.3
antibody from ascites fluid (1:500 dilution) which recognizes a 45,000-dahon protein specific for T
cells (9); (6) monoclonal 10.2 antibody from ascites fluid (1:500 dilution) which recognizes a 65,000-
67,000 dalton protein specific for T ceils (11); and (7) monoclonal 9.6 antibody from ascites fluid
(1:500 dilution). Immune precipitates were collected on S. aureus, eluted in electrophoresis buffer
that contained 2-mercaptoethanol, and analyzed by SDS-PAGE. Radioautography of the dried gel
was enhanced for 2 d on x-ray intensifying screens.
antigen is either identical to or closely associated with the E receptor because of (a)
the high degree of concordance between E-rosette formation and 9.6 antigen expres-
sion, (b) the inhibition of rosette formation by preincubation of cells with 9.6 antibody,
and (c) the observed failure of cells lysostripped of 9.6 antigen to form E-rosettes. This
last finding suggests cocapping of 9.6 antigen and the E receptor.
Received for publication 1 August 1980 and in revised form 3 November 1980.
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... The T-cell antigen CD2 (T il, LFA-2) was first identified as the sheep erythrocyte receptor (Kamoun et al., 1981). CD2 appears early during foetal development (day 13) and is expressed on >95% of adult thymocytes and peripheral T-cells (Kyewski et al., 1989a;Altevogt et al., 1989). ...
Thesis
This thesis is directed towards two separate but closely related goals; one involves the biology of precursor cells in T-cell development and the second involves the use of retroviral constructs for the establishment of thymic stromal cell lines and its application for studying differentiation of T-cells in vitro. Using the mouse foetal thymic organ culture system, a quantitative assay was devised in which the relative abundance of T-cell progenitors among different populations was estimated by seeding decreasing numbers of precursors into alymphoid thymic rudiments. This limiting dilution approach combined with serial transfer of successfully recolonised precursor cells in organ culture, provided evidence that certain populations seeding the thymus have the potential for extensive cell divisions (up to 10- 12 weeks). Stromal cells from adult and foetal sources were transformed by a temperature sensitive (ts) mutant of SV40 and Ela 12S in order to study interactions between the developing thymocytes and specific elements of the microenvironment. Eighty four cell lines were generated and have been shown to contain properties related to their counterparts in vivo. The thermolabile transforming agent (large T) allowed inactivation of the immortalising gene when the cells were switched to the nonpermissive temperature. Properties like the expression of MHC antigens and the ability to bind thymocytes could be induced by growing ts-derived clones at the nonpermissive temperature, which suggests that although cell proliferation had ceased, the cells were still metabolically active and had reverted to a more 'normal' non-transformed phenotype allowing certain molecules to be expressed at the cell surface. Close interaction of the developing thymocytes with the stroma is essential for the development of functionally mature T-cells. Using a rosette assay unfractionated adult thymocytes were found to bind unstimulated Ela-derived clones resembling epithelial cells (15.5 and 15.18) and also to several other established clones after temperature switching and/or IFNγ treatment. In co-culture with a multipotent bone-marrow derived stem cell clone (A4) it was shown that both proliferation and differentiation into certain myeloid lineages were supported. Furthermore, in co-culture with CD4'CD8' thymocytes, with and without interleukin-7, the growth of this subset could be sustained for ~20 days. In addition, phenotypic changes of thymocytes in these cultures suggest that some of these lines may have the potential to induce differentiation of early precursors. These findings indicate that established cell lines could be useful tools for studying maturation of T-cells in vitro and furthermore, to investigate distinct events in T-cell ontogeny governed by different stromal cell types.
... The CD2 molecule is one of the earliest antigens of the T cell lineage and is a 50 kd glycoprotein on the surface of all T cells and thymocytes (Howard et al, 1981;Kamoun et al, 1981). This molecule was originally found as the sheep erythrocyte receptor. ...
Thesis
The aim of this project was to analyse the tissue distribution and function of a novel gene cloned by Jenny Dunne within ICRF. Sequence data revealed that the gene structure is homologous to the EGF-like protein family in the arrangement of cysteine and other conserved amino acids. The cloned cDNA was truncated at both the N- and C-termini. To study the function, tissue distribution and the relationship with other proteins of this gene product, an antibody reacting with the native form of the gene product was considered to be essential. Several strategies have been followed in attempting to achieve this aim. Initially synthetic peptides derived from the gene sequence were used as immunogen, the reactivity of the polyclonal and monoclonal antibodies from immunised mice and rats did not correlate with the results of northern blot analysis and the antibodies did not react with native proteins. In a second series of experiments the truncated gene was linked in frame with a pseudo N-terminus and cloned into a vector which upon transfection can confer resistance to G418. The gene was stably integrated and transcribed. However, sera from mice immunised with the transfectants failed to stain living or fixed cells in a specific fashion. Surface iodination and internal labelling followed by one-D-gel analysis of cell associated or supernatant proteins did not show any difference between untransfected and transfected cells. This might suggest that the products were not processed onto the cell surface as expected. A third approach was to make several TrpE fusion proteins as immunogens. Antisera from rats showed no specific reactivity with the fusion proteins. The difficulty of raising antibody to the gene product is discussed. Northern blot analysis of various established lymphoid, myeloid, and epithelial cell lines, normal lymphoid tissues and activated lymphocytes showed that the gene is predominantly expressed in T-ALL cell lines. A striking feature of the northern analysis was the large number of different message sizes seen in different T cell lines. Cell surface phenotype analysis of these cells suggests that the pattern of transcripts is correlated with different stages of maturation. The implications of these findings for the function of this gene product and its possible role in malignant transformation are discussed.
Thesis
An analysis of the role of guanine nucleotide-binding proteins in T lymphocyte activation. Proteins which bind and hydrolyse GTP are involved in the regulation of many aspects of cellular growth control and metabolism. For example, the heterotrimeric G proteins are involved in transducing signals from receptors to effect changes in cellular second messenger systems. Another class of guanine nucleotide binding proteins (p21ras) are encoded by the ras protooncogenes and have been implicated in the regulation of normal cell growth and oncogenic transformation. Using a permeabilised cell system which allowed cellular access for otherwise membrane-impermeant nucleotide and peptide reagents, while maintaining many intracellular signalling pathways, the role of guanine nucleotide binding proteins in T cell antigen receptor (TCR/CD3) complex coupling to phospholipase C (PLC) and in the regulation of protein kinase C (PKC) activity were investigated. In addition, the regulation of p21ras by T cell surface receptors was analysed in both intact and permeabilised cells. The data show that the TCR/CD3 complex and a population of G proteins can regulate PI-PLC in T cells but that the effects of guanine nucleotides on TCR/CD3 coupling are not compatible with a simple receptor G protein PI-PLC model. These results most likely reflect that the TCR/CD3 complex is not coupled to PLC via a G protein but that a guanine nucleotide binding protein can indirectly modulate TCR/CD3 coupling. An alternative TCR/GD3 coupling mechanism is suggested. Receptor and G protein agonists were also observed to induce PKC- mediated phosphorylation of the CD3 y subunit in permeabilised T lymphoblasts. These results are consistent with stimulation of PKC activity being explained by agonist effects on phosphatidylinositol metabolism. However, the ability dissociate G protein agonist effects on PI-PLC activity and phosphorylation of CD3 suggest that guanine nucleotide binding proteins also exert a regulatory effect on PKC activity by distinct mechanisms. This study also demonstrates that the activation state of endogenous p21 can be regulated by triggering the TCR/CD3 complex, CD2 antigen or the high affinity receptor for the growth factor IL2. These results infer that normal p21ras functions in the signalling pathways by which these receptors regulate T lymphocyte activation and proliferation. Evidence is provided to suggest that the TCR/CD3 complex regulates the GTPase activity of p21ras and that both PKC-dependent and independent pathways exist for the regulation of p21ras in T lymphocytes.
Article
BTI-322, a rat monoclonal IgG2b directed against the CD2 antigen on T cells and natural killer (NK) cells, blocks primary and memory alloantigen proliferative responses in vitro. We have evaluated the pharmacokinetics and safety of BTI-322 during treatment of 20 transplant recipients with steroid-refractory acute graft-versus-host disease (GVHD). Treatment consisted of BTI-322 by intravenous (IV) bolus or 30-minute infusion at approximately 0.1 mg/kg/d for 10 days in addition to continuing high-dose steroids and tacrolimus or cyclosporine. Pharmacokinetic sampling was performed in 10 patients; the t1/2 ± SE was 9.1 ± 1.3 hours, the Cmaxwas 2,549 ± 291 ng/mL, the Vd was 3.97 ± 0.95 L, and the Vd/kg was 0.05 ± 0.01 L/kg. Ten patients experienced transient dyspnea sometimes accompanied by nausea, vomiting, diarrhea, and tachycardia shortly after the initial bolus dose of drug, but serious drug-related adverse events were not seen during the remainder of the infusions. At the end of treatment (day 11), there were six patients with complete responses and five with a reduction in grade of GVHD for a total response rate of 55% (95% confidence interval [CI], 32% to 77%). Antibodies targeting CD2 may be active in the treatment of acute GVHD, and evaluation of a humanized form of BTI-322 is warranted.
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The pathogenesis of many immune-mediated diseases of the urinary tract has been well elucidated in humans and in their respective animal models. While laboratory animals develop spontaneous immune-mediated renal disease, far less is known about their etiology or pathogenesis. By examining the five types of hypersensitivity reactions that lead to immune-mediated glomerular disease in humans, and understanding the molecular pathogenesis of lesion development, toxicologic pathologists can interpret test article-related lesions in the urinary tract, and help distinguish these lesions from spontaneous disease. Resident macrophages and dendritic cells of the kidney maintain continual tolerance, and any disruption of this homeostasis will result in glomerular and/or tubulo-interstitial inflammation which often self-perpetuates leading to end stage renal disease. Although immune dysfunction leads to renal disease, the reverse is true as well. Uremia leads to immunodysfunction, hypercytokinemia, and multi-organ damage especially to the cardiovascular system.
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Imaging with In-111-labeled leukocytes has become an established clinical method for the detection of focal inflammatory lesions in many medical centers but has been rejected by others because of the technical complexities of the labeling procedure. Gaining knowledge of the in vivo migratory pattern of the different leukocyte populations and subtypes in health and disease remains an important goal(1), particularly in the field of immunology. Techniques for examining the in vivo distribution of lymphocytes, monocytes and the eosinophils are still under development, and differences in migratory patterns of mononuclear subtypes remain to be explored. This paper attempts to summarize recent progress in the techniques of harvesting and labeling leukocytes and suggests possible directions for future research.
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Radioiodinated proteins have been used extensively for studying the rate of degradation of serum proteins in experimental and clinical studies (see Additional Readings, Chapter 12).
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We shall first briefly review the reactivity on normal lymphoid cells of various monoclonal antibodies we have used to study malignant tumor cell populations. We then emphasize the most important technical requirements for assessing the reactivity of MA with cell surfaces. Finally we report our results from the investigation of 66 cases of T-cell malignancy and of 111 cases of B and non-B, non T cell malignancies.
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By using the two criteria (a) high density of immunoglobulin determinants on the cell surface and (b) presence of receptors for C'3 on the cell surface for defining bone marrow-derived lymphocytes, it is indirectly shown that all or at least a major population of human thymus-derived lymphocytes under certain conditions will form nonimmune rosettes with sheep red blood cells (SRBC). Almost all thymocytes tested from two different donors formed rosettes. The SRBC rosettes are not formed by virtue of immunoglobulin receptors and form only around living cells. Positive bivalent ions are required for rosette formation since EDTA will block rosette formation. Sodium iodoacetate will also block rosette formation demonstrating the dependence on an intact glycolytic pathway. Rosette formation is temperature dependent and will not appear at 37°C. Trypsin treatment of lymphocytes will abolish their SRBC-binding ability which cannot be restored by treating them with fresh donor serum or fetal calf serum, but which will reappear after culturing the lymphocytes. It is suggested that these rosettes are formed by a rapidly released or metabolized receptor substance on the living cell surface which behaves as a trypsin-sensitive structure produced by the cells themselves.
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Long-term growth (now over 13 months) of thymus-derived lymphocytes from numerous normal human bone marrow and peripheral blood cell samples was accomplished by using a factor present in media obtained from mitogen-stimulated human peripheral blood lymphocytes. This long-term growth could neither be initiated nor maintained by mitogens alone. All cell cultures were greater than 90% E rosette-positive, whereas the tests for B cell markers, surface IgG and IgM, and EAC rosette were routinely negative. There was no evidence for the presence of granulocytes, monocytes, and their precursors in these cultures. The E rosette-positive cells were then tested to see if they had T cell functions. PHA, Con A, and pokeweed mitogens stimulated lymphproliferative responses in these cultures comparable to those of fresh peripheral blood cells. These proliferating cells were also able to release cell mediators, such as interferon and colony-stimulating activity. Further evidence for the T lymphocyte nature of these cultured cells was obtained from one-way mixed leukocyte cultures in which these cells responded to but were unable to stimulate allogeneic cells. The functional and morphologic characteristics of these cultured cells show that these cells are T cells that grow continuously in vitro.
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A series of mouse hybridomas producing monoclonal antibodies against human acute lymphocytic leukemia (ALL) cells was generated and screened for tumor specificity. Among 1200 primary cultures, 60 produced an antibody that could distinguish between the immunizing leukemia cells and an isologous B lymphoblastoid cell line. Of these, two produced an antibody that detects an antigen expressed preferentially on ALL cells and on a subpopulation of normal cells found in the cortex of the thymus. Other normal human lymphoid cells from lymph nodes, spleen, bone marrow, and peripheral blood express only low levels of this antigen. High levels of this "thymus-leukemia" antigen were found on T-ALL cells, T-ALL-derived cell lines, and some "null" ALL cells. By contrast, B-cell leukemias, B lymphoblastoid cell lines, and normal and malignant myeloid cells contain either low or undetectable amounts of this antigen. The thymus-leukemia antigen has been isolated from the membranes of leukemia cells by detergent solubilization and subsequent immunoprecipitation with the monoclonal antibody. Preliminary biochemical characterization shows the antigen to be associated with a polypeptide of Mr approximately 28,000.
Article
Multiple lymphoid cell lines were derived from 35HLA-D homozygous donors by EB-viral transformation of B lymphocytes. The expression of Ia-like alloantigens (HLA-DR) was studied by microcytotoxicity and by absorption with alloantisera exchanged through the Seventh Histocompatibility Workshop. B-lymphoid lines expressed the same specificities as normal B lymphocytes. Workshop antisera representing DRw1, DRw2, DRw3, and DRw7 gave well-defined typing patterns with cell lines derived from donors of corresponding D-locus specificities. A more complex reaction pattern was seen for antisera representing DRw4, DRw5, and DRw6. The available reagents could not discriminate between lines from donors homozygous for Dw4, Dw10, or D-KH. All lines studied, except for those from one donor homozygous for a unique D-locus determinant (D-SPO), could be assigned one of the provisional DRw specificities. The advantage of obtaining multiple cell lines from a single donor was evident. One line could not be typed by microcytotoxicity because it was lysed in all human sera tested, and some other lines gave weak cytotoxic reactions. Absorption studies, however, did indicate similar expression of DRw antigens on these lines. The availability of multiple lines from the same donor circumvented these difficulties.
Article
A new genetically polymorphic cell surface antigen recognized by a monoclonal rat anti-mouse antibody is expressed on mouse lymphoid cells. Fluorescence analysis on the fluorescence-activated cell sorter (FACS) locates the antigen on thymocytes, lymph node cells, and both T and B cells in the spleen. It also appears on approximately 40% of cells in the bone marrow. Immune precipitations from surface iodinated spleen cells followed by 2-D gel electrophoresis demonstrate that the antigen is a glycoprotein of approximately 100,000 daltons. Since it is expressed in all lymphoid tissues and on both T and B cells, we designate it lymphoid glycoprotein 100 (Lgp100). Strains with Lgp100 include A/J, AKR/J, AKR/Cu, BALB/c, 129/J, CBA/N, C3H/HeJ, CBA/2J, and SJL/J. Strains with no detectable antigen include C57BL/6J, C57BL/10J, C57BR/cdJ, C57L/J, and C58/J. Intercrosses and backcrosses establish a pair of alleles, a positive and a negative one, at a single locus. Heterozygotes display about 50% as much antigen as homozygotes by quantitative membrane immunofluorescence on the FACS. Tests for Lgp100 in 35 recombinant inbred strains from three crosses (CXB, BXB, and BXH) locate this locus on chromosome 1, closely linked to theMls locus.
Article
We describe here two new monoclonal antibodies that react with surface antigens of human lymphocytes. Antibody 7.2 identified a determinant on the framework region of the human Ia antigen. It was cytotoxic for all cultured B-cell lines, normal B cells, and monocytes. The antibody was not cytotoxic for normal T cells or for established T leukemic cell lines. In immune precipitation assays, the 7.2 antibody reacted with a bimolecular complex of two chains that resolved in polyacrylamide gels as polypeptides with molecular weights of 29000 and 34000 daltons. These precipitation results were analogous to those achieved with a rabbit antiserum prepared against human Ia antigens. Antibody 9.3 identified a determinant on the framework region of a T-cell antigen. It was cytotoxic for 50–80% of peripheral T cells and for 20–50% of thymocytes. It was not cytotoxic for cultured B-cell lines, normal B cells, or monocytes. In immune precipitation assays, the 9.3 antibody reacted with a single polypeptide with a molecular weight of 44000 daltons. Due to the expression of this antigen on a limited subpopulation of human T cells, we have designated the antigen HuLyt-1.
Article
A monoclonal antibody directed at a determinant on human peripheral blood T cells was produced and characterized. This hybridoma antibody, termed OKT1, was reactive by indirect immunofluorescence with the entire human peripheral blood T cell population and a subset of human thymocytes. In contrast, OKT1 was unreactive with normal B cells, Null cells, macrophages, and ≥90% of human thymocytes. These findings suggested that OKT1 defines a mature T cell differentiation antigen. In support of this notion was the observation that T cell acute lymphoblastic leukemia cells were nonreactive with OKT1, whereas T cell chronic lymphocytic leukemia cells were reactive. Concomitant functional studies on FACS-separated lymphocytes showed that the T cell proliferative responses to mitogens and soluble and cell surface antigens were contained in the OKT1+ population. Fractionation of peripheral blood T cells into strongly and weakly reactive OKT1+ subgroups uncovered no functional T cell heterogeneity. In addition, when the thymocyte population was separated into OKT1+ and OKT1- subsets, only the OKT1+ thymocytes were MLC responsive. However, unlike peripheral T cells, neither the OKT1+ or OKT1- thymocytes proliferated to the mitogens PHA and Con A. Thus, OKT1+ thymocytes are functionally distinct from OKT1+ peripheral blood T cells. These studies show that a hybridoma antibody can be produced that detects a human differentiation antigen that appears during late intrathymic T cell ontogeny and persists on peripheral T cells.
Article
Three novel nonoclonal antibodies (designed OKT1, OKT3, and OKT4) were generated against surface determinants of human peripheral T cells. Both OKT1 and OKT3 reacted with all human peripheral T cells and 5 to 10 percent of thymocytes but differed in their reactivities with T cel- lines. By contrast, OKT4 reacted with 55 percent of human peripheral T cells and 80 percent of thymocyted in that they did not react with normal B cells, null cells, monocytes, or granulocytes.
Article
Spleen cells from a BALB/c mouse that had been immunized with human thymocytes were fused with the myeloma line P3-NS 1/1 Ag 4.1. One of the resulting hybrid clones (NA 1/34) secreted an antibody that was highly specific for human thymocytes. Eighty-five % of thymocytes expressed the antigen designated HTA1. There were an estimated 15 x 10(4) molecules of HTA 1 per cell, and it is therefore a major surface molecule. The expression of this antigen on thymocytes appears to be reciprocal to HLA, as recognized by another monoclonal antibody W6/32. Immunoprecipitated material from [125I]-labeled thymocyte membranes was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate which disclosed a single component of 45,000 molecular weight.