Download Identification of a T cell surface molecule using a monoclonal

 Original article
Identification of a T cell surface molecule using a
monoclonal antibody produced by TCR/CD3 complex
immunization
Kodchakorn Mahasongkram,1 Supansa Pata,1,2 Nuttapol Chruewkamlow1 and Watchara Kasinrerk1,2
Summary
Background: Several molecules are known to be
involved in T-cell activation via the TCR/CD3
complex and while the mechanisms of late T cell
signaling have been well characterized, the very
early events are still not fully understood.
Objective: The aim of this study was to identify
yet unknown molecules associated with the
TCR/CD3 complex.
Results: To identify new molecules associated
with the TCR/CD3 complex, a monoclonal
antibody termed MT3 was produced by
immunoprecipitated
beads
immunization.
Colocalization of the MT3 mAb recognizing
molecules with the TCR/CD3 complexes was
verified by confocal microscopic analysis. The
surface antigen recognized by MT3 antibody was
expressed on a subpopulation of CD3+ T cells,
and on both CD4+ and CD8+ lymphocytes. The
antigen was also expressed on naïve CD4+ T cells
and on a subset of memory CD4+ T cells. In
contrast, in the CD8 population, the majority of
MT3+ cells were found in the naïve population.
The MT3 mAb recognizing molecules were also
expressed on red blood cells but only in
particular subjects. Similar to peripheral blood
leukocytes, MT3 mAb recognizing molecules are
exclusively expressed on T cell lines.
From 1. Division of Clinical Immunology, Department of
Medical Technology, Faculty of Associated Medical
Sciences, Chiang Mai University, Chiang Mai 50200,
Thailand
2. Biomedical Technology Research Center, National
Center for Genetic Engineering and Biotechnology,
National Science and Technology Development Agency at
the Faculty of Associated Medical Sciences, Chiang Mai
University, Chiang Mai 50200, Thailand
Corresponding author: Watchara Kasinrerk
E-mail: [email protected]
Submitted date: 1/7/2014
Accepted date: 14/10/2014
Conclusions: Based on the cellular distribution
patterns and confocal microscopic analysis, the
MT3 mAb recognizing molecule that we
investigated is proposed to be a TCR/CD3
associated molecule and might be involved in the
antigen recognition of T cells. (Asian Pac J Allergy
Immunol 2015;33:107-16)
Keywords:
TCR/CD3
complex,
hybridoma,
immunoprecipitation, leukocyte surface molecule, T
cell activation
Introduction
T cell activation is a crucial step for initiating
adaptive immune responses.1-3 Initiation of T cell
activation requires extracellular ligation of the T cell
receptor (TCR) with antigen presented by antigenpresenting cells, and the subsequent formation of
dynamic signaling complexes.2,4-6 On T cells
surface, the TCR is expressed as a complex with
CD3 molecules, TCR/CD3 complex, to convey
intracellular signals.2 Upon TCR/CD3 triggering,
several other molecules are required for full-fledged
T cell activation.7-11 Although the mechanisms of T
cell activation inside T cells have been well
characterized, evidence suggests that that new or
not-so-well-defined molecules might be associated
to TCR/CD3 complexes and involved in the
TCR/CD3
activation
and
need
further
characterization.
Recently,
the
immunoprecipitated
bead
immunization was established in our laboratory in
order to effectively produce monoclonal antibody
(mAb) of interest.12 By this technique, mAbs
specific to the molecule of interest are coated on
beads and used to precipitate protein of interest in
the samples. The antigen bound immunoprecipiated
beads are, then, directly used for inoculating mouse.
Using this strategy, we have produced several mAbs
of interests.12,13
In order to identify new/yet unknown molecules
associated with TCR/CD3 complex which will lead
to a better understanding of the early events after
TCR engagement, the immunoprecipitated beads
107 Asian Pac J Allergy Immunol 2015;33:107-16 DOI 10.12932/AP0538.33.2.2015
immunization strategy was employed. In this study,
anti-CD3 mAb coated beads were used to precipitate
the TCR/CD3 complex from the human peripheral
blood mononuclear cell lysate. We presumed that
the putative TCR/CD3 associated molecules would
be co-precipitated and thus bound on the
immunoprecipitated beads. The immunoprecipitated
beads were then used as an immunogen for the mAb
production. By this approach, a mAb named MT3
which reacted to a sub-population of T lymphocytes
and colocalized with TCR/CD3 molecules, was
obtained. From the cellular distribution pattern, the
MT3 mAb recognizing molecule might be an undefined molecule which associate to the TCR/CD3
complex and might play an important role in T cell
activation.
Methods
Cells, cell lines, cell lysates and Reagent
Peripheral blood mononuclear cells (PBMCs)
were isolated from EDTA blood by standard FicollHypaque gradient centrifugation method using
Ficoll-Hypaque solution (GE Healthcare Life
Sciences, Uppsala, Sweden). Cell lines including
SupT1, Molt4, Jurkat, HuT78, HPB-ALL, Daudi,
Ramos, Raji, U937, THP-1, HL-60, K562, and KG1a were used in this study. All the cell lines were
maintained in RPMI-1640 medium (GibcoInvitrogen, Grand Island, NY, USA) supplemented
with 10% fetal bovine serum (FBS; GibcoInvitrogen), 40 μg/ml gentamycin, and 2.5 μg/ml
amphotericin B (10%FBS-RPMI) in a 5%
CO2 incubator at 37°C.
For preparation of cell lysates, PBMCs (5x107
cells) were solubilized in 1 ml of lysis buffer (1%
Brij-58, 50 mM Tris-HCl pH 8.2, 100 mM NaCl, 2
mM EDTA, 1 mM PMSF, 5 mM Iodoacetamide, 10
µg/ml aprotinin) for 30 min on ice. Cell lysate was
clarified by centrifugation at 12,000 rpm for 30 min
at 4°C.
Anti-CD99 (MT99/3; IgM isotype),14 antiCD147 (M6-1D4; IgM isotype)15 and antihemoglobin (Hb1a; IgM isotype) were produced in
our laboratory. Anti-CD14 (VIM-13; IgM isotype)
mAb was kindly provided by Prof. Vaclav Horejsi
(Institute of Molecular Genetics, Academy of
Sciences of the Czech Republic, Prague, Czech
Republic). PerCP-Anti-CD3 (SK7), PE-anti-CD4
(SK3), PE-anti-CD8 (SK1) mAbs were purchased
from BD Biosciences (San Jose, CA, USA). PECy7A-anti-CD3 (UCHT1), PB-anti-CD4 (SK3),
PerCP-anti-CD8 (SK1) and FITC-anti-CD45RA
(HI100) mAbs were purchased from BioLegend
(San Diego, CA, USA). PE-anti-CD56 (MEM-188,
Caltag Laboratories, Burlingame, CA, USA), PEanti-CD19 mAbs (HIB19, eBioscience, San Diego,
CA, USA), FITC-anti-mouse IgM (µ chain specific)
Ab (Beckman coulter, Marseille, France), PE-antimouse IgM (µ chain specific) Ab (Beckman coulter,
Marseille, France) and APC-anti-mouse IgM (µ
chain specific) Ab (Jackson ImmunoResearch, West
Grove, PA, USA) were used in the flow cytometric
experiments. Alexa 568-labeled goat anti-mouse
IgG and Alexa 488-labeled goat anti-mouse IgM Ab
were purchased from Molecular Probes-Invitrogen.
Production of monoclonal antibody to molecule
associated with TCR/CD3 complex
A Hybridoma line producing mAb reacting with
molecule associated with TCR/CD3 complex was
established using the TCR/CD3 immunoprecipitated
beads immunization technique.12,13 In brief, TCR/CD3
complexes contained in PBMC lysates were
precipitated using anti-CD3 mAb (OKT3) coated
CNBr sepharose beads. A BALB/c mouse was
immunized with the immunoprecipitated beads three
times at 2-weeks interval. For beads immunization,
500 µl of beads in PBS suspension were
intraperitoneally immunized. The immunizations were
repeated following the same procedure 2 more times
at 2-week interval. Five days after the last boosting,
the mouse was sacrificed; splenocytes were
collected and fused with P3-X63Ag8.653 myeloma
cells by standard hybridoma technique using 50%
polyethylene glycol. After HAT medium selection,
the culture supernatants obtained from hybridoma
containing wells were analyzed for antibody
reactivity by indirect immunofluorescence analysis
using PBMCs as antigens. A hybridoma clone,
named MT3, which produces the mAb MT3 that
reacts to a population of lymphocytes, was selected
and subcloned by limiting dilution. The isotype of
the mAb MT3 was determined using an isotyping
ELISA kit (Sigma-Aldrich, St. Louis, MO, USA).
The institutional guidelines were followed for the
care and use of the animals.
The hybridoma clone MT3 was cultured in
serum-free medium (Gibco-Invitrogen) containing
penicillin (100 U/ml) and streptomycin (100 µg/ml)
for the large-scale production of mAb. The mAb
MT3 was purified from the culture supernatants by
using HiTrapTMIgM column (GE healthcare Life
sciences). The purified mAb MT3 was then checked
for its purity by SDS-polyacrylamide gel
108 Identification of a molecule associated with TCR/CD3 electrophoresis (SDS-PAGE) and its reactivity by
indirect immunofluorescence.
Flow cytometric analysis
For lysed whole blood staining, EDTA bloods
were stained with 30 µg/ml of the mAb MT3 or with
the control mAbs for 30 min at room temperature
(RT). After washing, the cells were stained with PE
conjugated anti-mouse IgM (µ chain specific) Ab
for 30 min at RT. Red blood cells were then lysed
by using FACS lysis solution (BD Biosciences) for
15 min in the dark at RT. The stained cells were
washed twice and analyzed using a FACSsort flow
cytometer (BD Biosciences).
For red blood cells (RBCs) staining, EDTA
blood were centrifuged at 12,000 g for 1 minute and
washed with PBS for 3 times. The packed RBCs
were collected and adjusted to 0.3% cells in PBS
containing 1% BSA and 0.02% NaN3 (1%BSAPBS-0.02%NaN3). The cells were stained with 30
µg/ml of the mAb MT3 or with the control mAbs
for 30 min at 4°C. After washing, the bound mAbs
were detected by counter staining with the FITC
conjugated anti-mouse IgM (µ chain specific) Ab
for 30 min at 4°C. The stained RBCs were gated and
analyzed using a FACSsort flow cytometer (BD
Biosciences).
For PBMCs and cell lines, the cells were stained
with 30 µg/ml of the mAb MT3 or with the control
mAbs for 30 min at 4°C. After washing, the bound
mAbs were detected by counter staining with the
FITC conjugated anti-mouse IgM (µchain specific)
Ab for 30 min at 4°C. The stained cells were
analyzed using a FACSsort flow cytometer (BD
Biosciences).
For the two-color flow cytometric analysis, the
PBMCs were incubated with 30 µg/ml of the mAb
MT3 and PE-anti-CD3, or PE-anti-CD19, or PEanti-CD56 mAbs for 30 min at 4°C. The bound
mAb MT3 was detected by staining with the FITCanti-mouse IgM (µ chain specific) Ab for 30 min at
4°C. The stained cells were analyzed by using a
FACSsort flow cytometer (BD Biosciences).
For the three-color flow cytometric analysis,
PBMCs were stained with the mAbs MT3 (30
µg/ml) and the PerCP-anti-CD3, or PE-anti-CD4, or
PE-anti-CD8 mAbs. The mAb MT3 was detected
with the FITC-anti-mouse IgM (µ chain specific)
Ab. Upon flow cytometric analysis, the CD3+ T
cells were gated and the distributions of MT3 mAb
recognizing molecules in CD4+ and CD8+ T cells
were determined.
For the five-color flow cytometric analysis,
PBMCs were stained with the mAbs MT3 (30
µg/ml) and the PE-Cy7A-anti-CD3, FITC-antiCD45RA, perCP-anti-CD8, and PB-anti-CD4
mAbs. The mAb MT3 was detected using the APCanti-mouse IgM (µ chain specific) Ab. Upon flow
cytometric analysis, the CD3+ T cells were gated.
The CD4+ and CD8+ T cells were then gated and the
distribution of MT3 mAb recognizing molecules in
naïve and memory CD4+ and CD8+ T cells were
determined.
Confocal Microscopic analysis
PBMCs or Jurkat cells were stained with the
mAb MT3 (30 µg/ml) and anti-CD3 mAb (OKT3;
20 µg/ml) at 4°C for 30 min. The stained cells were
washed, and incubated with Alexa 568-labeled goat
anti-mouse IgG and Alexa 488-labeled goat antimouse IgM Ab at 4°C for 30 min. The stained cells
were fixed for 10 min in 4% formaldehyde-PBS at
RT and plated on a poly-D-lysine-coated slide. The
images were acquired on a confocal microscope
(Nikon, Tokyo, Japan).
Expression of MT3 mAb recognizing molecules
upon PBMC activation
PBMCs (5×105cells/ml) were either unstimulated
or stimulated with immobilized anti-CD3 mAb
(OKT3; 60 ng/ml) or 10 ng/ml PMA and 1 μM
ionomycin for 3 days in a 5% CO2 incubator at
37°C. The activated cells were harvested and stained
with 30 µg/ml of mAb MT3 for 30 min at 4°C and
PE conjugated anti-mouse IgM (µ chain specific)
Ab at 4°C for 30 min. The expression of MT3 mAb
recognizing molecules on lymphocytes was
determined by FACSsort (BD Biosciences).
Ethics
The study was approved by the ethical and
scientific committees of the Faculty of Associated
Medical Science, Chiang Mai University. For
experiment in animals, the institutional guidelines
were followed for the care and use of the animals.
Results
Production of monoclonal antibody to TCR/CD3
associated molecule
To produce mAb to TCR/CD3 associated
molecule, in this study, the immunoprecipitated
beads immunization technique was performed. AntiCD3 mAb coated beads were used to precipitate
TCR/CD3 complexes and its associated molecules.
The immunoprecipitated beads were, then, used for
mouse immunization. Splenocytes from the
109 Asian Pac J Allergy Immunol 2015;33:107-16 DOI 10.12932/AP0538.33.2.2015
immunized mouse were then fused with mouse
myeloma cells by standard hybridoma technique.
Specificity of the obtained mAb produced by the
generated hybridomas was examined by indirect
immunofluorescent staining using PBMCs as target
cells. By this screening procedure, a hybridoma
clone produced mAb which selectively reacted with
subpopulation of lymphocytes was chosen for
further analysis (data not shown). This mAb was
designated as MT3 and has IgM isotype.
Cellular expression of MT3 mAb recognizing
molecules on peripheral blood cells
To determine the expression of molecule
recognized by mAb MT3 (MT3 mAb recognizing
molecule) on human leukocytes, whole blood
staining was performed. It was found that the mAb
MT3 reacted with a subpopulation of lymphocytes,
but did not react to granulocytes and monocytes
(data not shown).
The subpopulations of lymphocyte which express
the MT3 mAb recognizing molecules were further
investigated by two color flow cytometric analysis.
Within the lymphocyte population, the mAb MT3
selectively reacted to a subpopulation of CD3+ T
cells, but did not react to CD19+ B cells and CD56+
NK cells (Figure 1A). We then determined the
expression of MT3 mAb recognizing molecules on
subsets of CD3+ T cells, CD4+ and CD8+ cells. Three
color flow cytometric analysis revealed that MT3
mAb recognizing molecule expressed on a
subpopulation of CD4+ (mean ± SD; 42.13±3.68%)
and CD8+ T cells (mean ± SD; 21.14±1.96%; Figure
1B).
The expression of MT3 mAb recognizing
molecules on naïve and memory T cell subsets was
also determined by five-color flow cytometric
analysis. The results indicated that MT3 mAb
recognizing molecules were differentially expressed
on naïve and memory CD4+ and CD8+ T cell
subsets. MT3 mAb recognizing molecules on all
CD4+CD45RA+ naïve T cells and also on a subset of
CD4+CD45RA- memory T cells (Figure 1C). In
contrast, in the CD8 subset the majority of MT3+
cells were found in the naïve (CD45RA+)
subpopulation, CD8+CD45RA+ T cells (Figure 1C).
In addition, we analyzed the expression of MT3
mAb recognizing molecules on RBCs. Interestingly,
we found that MT3 mAb recognizing molecule
heterogeneously expressed on RBCs. About 60% of
tested RBCs’ donors were reactive with mAb MT3
(Figure 2). No relation between MT3 mAb
recognizing molecule expression on RBC and ABO
blood group of the donors was found (data not
shown).
Expression of the MT3 mAb recognizing molecules
on hematopoietic cell lines
To determine the expression of MT3 mAb
recognizing molecules on hematopoietic cell lines,
various cell lines were stained with mAb MT3 or
isotype matched control mAb. Similar to the results
obtained with peripheral blood leukocytes, mAb
MT3 specifically only reacted with to T cell lines
(Figure 3A). Within the T cell lines, the MT3 mAb
recognizing molecule was strongly expressed on
SupT1 and Molt4 T cell lines, whereas expression
was low on Jurkat, HuT78 and HPB-ALL cells
(Figure 3A).
Colocalization of MT3 mAb recognizing molecules
and CD3 molecules on cell surface
As mAb MT3 was produced by using the
TCR/CD3 complex as the immunogen, we
speculated that the MT3 mAb recognizing
molecules were associated with the TCR/CD3
complexes. To test this assumption, colocalization
of the MT3 mAb recognizing molecules and CD3
molecules was performed and the results were
visualized
using
confocal
microscopy.
Colocalization of the MT3 mAb recognizing
molecules and CD3 molecules was observed in
peripheral T cells and also in the Jurkat T cell line
(Figure 4 and 5, respectively). As shown in Figure
6A, the MT3 mAb recognizing molecules and CD3
molecules were clearly colocalized on T cell
surface. However, in some T cells, not all of the
MT3 mAb recognizing molecules and CD3
molecules were colocallized, as a few molecules
were found to be expressed in free form as well as in
Jurkat cell line (Figure 4B and 5).
Down-regulation of MT3 mAb recognizing
molecules upon T cell activation
To determine the alteration of the expression of
MT3 mAb recognizing molecules in resting and
activated T cells, PBMCs were stimulated with or
without immobilized anti-CD3 mAb (OKT3) or
PMA and ionomycin. The MT3 mAb recognizing
molecules on cell surface determined by flow
cytometry. The expression of MT3 mAb
recognizing molecules was down-regulated upon T
cell activation (Figure 6).
110 Identification of a molecule associated with TCR/CD3 Figure 1. Expression of MT3 mAb recognizing molecules on human T cell subsets. (A) PBMCs were stained with
the mAbs MT3 and the PerCP-anti-CD3, or PE-anti-CD19, or PE-anti-CD56 mAbs. The mAb MT3 was detected
with the FITC-anti-mouse IgM (μ chain specific) Ab. The lymphocytes population was gated and the expression of
MT3 mAb recognizing molecules in each subset are shown. The results are representative of four healthy
individuals. (B) PBMCs were stained with the mAbs MT3 and the PerCP-anti-CD3, or PE-anti-CD4, or anti-CD8
mAbs. The mAb MT3 was detected with the FITC-anti-mouse IgM (μ chain specific) Ab. The CD3+ T cells were
gated and the distributions of MT3 mAb recognizing molecules in CD4+ and CD8+ T cells are shown in the dot
plot. The results are representative of four healthy individuals. (C) PBMCs were stained with the mAbs MT3, PECy7A-anti-CD3, FITC-anti-CD45RA, PerCP-anti-CD8, and PB-anti-CD4 mAbs. The mAb MT3 was detected
using the APC-anti-mouse IgM (μ chain specific) Ab. The CD4+ and CD8+ T cells were gated and the distribution
of MT3 mAb recognizing molecules in naïve and memory CD4+ and CD8+ T cells are shown in the plot. The
results are representative of three healthy individuals.
111 Asian Pac J Allergy Immunol 2015;33:107-16 DOI 10.12932/AP0538.33.2.2015
Figure 2. Expression of the MT3 mAb recognizing molecules on red blood cells. (A) Red blood cells were stained
with mAb MT3 (bold line) or IgM isotype matched control antibody (Hb1a, thin line). The bound mAbs were
detected by FITC-anti-mouse IgM (μ chain specific) Ab and assessed by flow cytometry. Red blood cells from ten
individual healthy donors were tested. (N represents the donor number).
Figure 3. Expression of MT3 mAb recognizing molecules on hematopoietic cell lines. Several T cell lines (A)
and non T cell lines (B) were stained with mAb MT3 or IgM isotype matched control antibody (VIM-13). The
bound mAbs were detected by APC-anti-mouse IgM (μ chain specific) Ab and assessed by flow cytometry. The
reactivity of mAb MT3 and isotype matched control antibody are represented in grey and open histogram,
respectively.
112 Identification of a molecule associated with TCR/CD3 Figure 4. Colocalization of MT3 mAb recognizing molecules and CD3 molecules on peripheral T cells. The PBMCs
were stained with the anti-CD3 mAb (OKT3) and the mAb MT3. The bound antibodies were then stained with Alexa
488-anti-mouse IgM μ chain specific and Alexa 568-anti-mouse IgG Abs. The colocalization of MT3 mAb
recognizing molecules and CD3 molecules was investigated under confocal microscopy. The confocal microscopy
detection of MT3 (green) and CD3 (red) on the cells surface are shown. TD represents the transmitted light channel.
Colocalization of MT3 mAb recognizing molecules and CD3 molecules are shown in the overlay image.
Discussion
Monoclonal antibodies are an important tool in
many applications, including biological research,
medical diagnostics and therapy.16-21 Although the
hybridoma technique has been discovered almost 40
years ago,22 it is still the standard method for
production of monoclonal antibodies. Recently, for
enhancing the efficiency of the conventional
hybridoma technique, we have established an
immunoprecipitated bead immunization strategy for
mouse immunization prior to hybridoma technique.12
The
established
method
combines
the
immunoprecipitation
technique
and
bead
immunization. The immunoprecipitation technique
involves the interaction between a protein of interest
and its specific antibody coated on beads. After
immunoprecipitation, the immunoprecipitated beads
are directly used, without an eluting process, for
mouse immunization. By this strategy, antibodies
specific to protein of interest was satisfactorily
induced in the immunized mice. The spleen cells of
the immunized mice could also be used to generate
hybridomas producing mAb of interest.12,13 The
advantage of this immunoprecipitated bead
immunization is that the conjugation of antigen to
particulate beads transforms the soluble protein
antigen to the particulate antigen. The particulate
immunogen stimulates phagocytic activity and
cytokine production of antigen presenting cells
better than soluble immunogen, resulting in
enhanced immune responses.23 This technique
provides a powerful approach for production of
mAbs to proteins that are low in abundance or are
difficult to purify.
In an attempt to identify an undefined membrane
protein which is associated with the TCR/CD3
complex, in this study, we applied the
immunoprecipitated bead immunization. Anti-CD3
mAb immobilized on beads were used to capture
TCR/CD3 complexes in PBMC lysate. We
presumed that the TCR/CD3 associated molecules
would be co-precipitated and bound on the
immunoprecipitated beads. The immunoprecipitated
beads were directly used as the immunizing agent
for production of mAb using standard hybridoma
technique. Our study resulted in obtaining a mAb
named MT3 which reacted to a subpopulation of T
lymphocytes.
In this study, the cellular expressions of MT3
antigen which are recognized by the produced mAb
113 Asian Pac J Allergy Immunol 2015;33:107-16 DOI 10.12932/AP0538.33.2.2015
Figure 5. Colocalization of MT3 mAb recognizing molecules and CD3 molecules on Jurkat T cell line. Jurkat cells
were stained with the anti-CD3 mAb (OKT3) and the mAb MT3. The bound antibodies were detected with Alexa
568-anti-mouse IgM and Alexa 488-anti-mouse IgG Abs. The confocal microscopy detection of MT3 mAb
recognizing molecules (green) and CD3 (red) on the cells surface is shown. TD represents the transmitted light
channel. Colocalization of MT3 mAb recognizing molecules and CD3 are shown in the overlay image.
MT3 are described. MT3 mAb recognizing
molecules were demonstrated to be specifically
expressed on a subpopulation of CD3+ T cells. They,
however, were not expressed on surface of other
leukocytes including granulocytes and monocytes, B
cells and NK cells. Within CD3+ T cells, the MT3
mAb recognizing molecule was expressed on
subsets of the CD4+ and CD8+ T cells. The MT3
mAb recognizing molecule was also expressed on
all naïve CD4+ T cells and on a subset of memory
CD4+ T cells. In contrast, the majority of MT3+ cells
were found in the naïve CD8+ T cells. We used
confocal microscopic analysis to assess the
hypothesis that MT3 mAb recognizing molecules
should be associated with TCR/CD3 on T cells
surface. Colocalization of the MT3 mAb
recognizing molecules with CD3 molecules was
indeed
demonstrated.
This
indicates
the
colocalization of MT3 mAb recognizing molecules
and TCR/CD3 complexes. As TCR/CD3 complexes
express on all T cells, it is surprising that the MT3
mAb recognizing molecules do not express on all T
cells but express only on a T cell subpopulation. We
hypothesize that the MT3 mAb recognizing
molecule may be associated with the TCR/CD3
complex in a particular subset of T cells and it
would be interesting to analyze the functional
consequences of this association.
The expression of MT3 mAb recognizing
molecules on RBCs was also verified. Surprisingly,
we also found MT3 mAb recognizing molecules on
RBCs surface. However, the expression of MT3
mAb recognizing molecule was demonstrated only
in some subjects (6 in 10 tested subjects) and was
not related to the ABO blood group. As TCR/CD3
complexes are antigen receptors and exclusively
express on T cells.24,25 The expression of MT3 mAb
recognizing molecules on RBCs indicated that MT3
mAb recognizing molecule expression also as
TCR/CD3 un-colocalized form. This interpretation
corroborate our observations made using confocal
microscopy which demonstrated that some MT3
mAb recognizing molecules were expressed as
TCR/CD3 un-colocalized form. To the best of our
knowledge, this cellular distribution pattern of cell
surface molecules has never been described before.
The elucidation of the function of MT3 mAb
recognizing molecules on RBCs was undertaken as
a new research question. As we do not have direct
evidence that the molecule on RBCs and
lymphocytes that recognized by mAb MT3 are the
same, it should be precised that the reaction of mAb
MT3 with red blood cells may come from the cross
reactivity of the mAbs.
The expression of MT3 mAb recognizing
molecules on various cell lines was determined.
Similar to the peripheral blood leukocytes, MT3
mAb recognizing molecule express on surface of all
T cell lines tested, but not on other hematopoietic
cell lines. Confocal microscopic analysis of Jurkat
cells indicated both MT3-TCR/CD3 colocalized
form as well as un-colocalized form. The results
confirmed the findings from primary peripheral
blood T cells.
Altogether, our results demonstrate the
identification of a new TCR/CD3 associated
molecule, named MT3. The immunoprecipitated
bead immunization strategy was instrumental in
characterizing this new molecule. The cellular
distribution patterns suggest that the MT3 mAb
114 Identification of a molecule associated with TCR/CD3 Figure 6. Down-regulation of MT3 mAb recognizing molecules upon T cell activation. PBMCs were stimulated with
or without immobilized mAb OKT3 (anti-CD3 mAb) or PMA and ionomycin or for 3 days. The cells were harvested
and stained with mAb MT3 and the expressions of the molecule recognized by mAb MT3 were assessed by flow
cytometry. The FACS histograms of un-stimulated cells, OKT3 activated cells and PMA/ ionomycin activated cells
were shown. The results shown are representative of three healthy subjects.
recognizing molecule is an undefined TCR/CD3
associated molecule, which is expressed as
associated and un-associated form. The function of
the MT3 mAb recognizing molecule that underlying
antigen recognition and TCR/CD3 activation is
under investigation.
5.
Acknowledgments
This study was supported by the NSTDA
Research Chair Grant, National Sciences and
Technology Development Agency (Thailand), The
Thailand Research Fund and the National Research
University Project under the Thailand Office of the
Higher Education Commission. Kodchakorn
Mahasongkram is a doctoral candidate in the Royal
Golden Jubilee Ph.D. program of The Thailand
Research Fund. We would like to thank Prof.
Hannes Stockiger, Prof. Peter Steinberger and Dr.
Ludith Leitner, Medical University of Vienna, for
their kind assistances in flow cytometric analysis,
suggestions and manuscript preparation.
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