Vol. 4 No.

2 2000

New Products

Anti-Biotin MicroBeads 2

Novel Blood Dendritic

Cell Antigens 3

MACS Products

Human and
Mouse Cytokine
Secretion Assays 5

Customer Report

Enrichment of
Plasmodium-infected
red blood cells 7

Purification of allergen-
specific T cells based on
IL-4 and IFN-γ secretion 9

Meeting Report

CD133 (AC133)
Workshop 2000 11

M iltenyi B iotec
 NEW PRODUCTS
Der MACS r,
In June of this year over 150 people
took the opportunity to exchange their
experiences with MACS on a Cytokine
Secretion Assay Day that was held in
London and organized by Miltenyi
Biotec’s UK office. This again showed
us how significant the interest is in
our tool for the isolation of antigen-
specific T cells and that‘s why this
MACS & more has a Secretion Assay
accent with an overview on various
applications (page 5) including our
first customer report on this subject
(page 9).
CD133 (AC133) 
ndi rrr
cs
There are hints that CD133
(AC133) may be expressed also on
developmentally early muscle and
neural cells. During this year‘s AC133
workshop, at the ISEH, various
exiting data was presented indicating
that the hematopoietic stem cell
marker seems to be a potent marker
for the identification of endothelial
precursor cells. These and other news
on recent AC133 results are given in
the meeting report on page 11.
Exrc r r:
T rf
On page 6 you will find a brain
tease challenge in the form of some
questions about the Secretion Assay.
Detailed answers are given on page
15 where you can check whether you
are right.
Hope you enjoy “brainy” reading
2 © 2000 Miltenyi Biotec
Anti-Biotin MicroBeads
for most sensitive
indirect labeling
MACS Anti-Biotin MicroBeads for
research applications are now available
for the isolation of cells labeled with any
biotinylated primary antibody or ligand.
Whenever direct MicroBeads are not
available, MACS Indirect MicroBeads are
the perfect tool for the isolation, purification
and enrichment of virtually any cell type.
They can be used with any primary antibody,
whether unconjugated, fluorochrome
conjugated or biotinylated. Streptavidin
MicroBeads are frequently used when
working with biotinylated primary antibodies
or ligands. They are the ideal choice for any
material expressing the selection marker at
a normal to high level. With the new Anti-
Biotin MicroBeads we provide a refined
biotin-system with even higher sensitivity.
Anti-Biotin MicroBeads do not react with
free biotin, which is often present in cell
culture media.
Anti-Biotin MicroBeads will be the first
choice whenever an enhanced magnetic
labeling is desired and fluorochrome labeling
of the selection marker should be avoided.
MACS Indirect MicroBeads:
Anti-FITC MicroBeads
Anti-FITC MultiSort Kit (releasable, for multi-parameter cell sorting)
Anti-PE MicroBeads
Anti-Biotin MicroBeads
Streptavidin MicroBeads
Goat Anti-Rabbit IgG (H+L) MicroBeads
Mouse Anti-Human IgG MicroBeads
Goat Anti-Mouse IgG (H+L) MicroBeads
Rat Anti-Mouse IgG1 MicroBeads
Rat Anti-Mouse IgG2a+b MicroBeads
Rat Anti-Mouse IgM MicroBeads
Goat Anti-Rat IgG (H+L) MicroBeads
Mouse Anti-Rat Kappa MicroBeads
They are ideal for most efficient separation
of cells with weakly expressed antigens. Anti-
Biotin MicroBeads also offer the advantage
of optimal magnetic labeling when working
with a cocktail of antibodies directed against
a combination of weakly and strongly
expressed markers. This is especially required
for depletion of several cell types in one step.
When using Anti-Biotin MicroBeads, MS,
LS or XS Columns are recommended for
positive selection. The use of these columns
will lead to highly pure positive fractions
and, in most cases, to highly pure negative
fractions as well. However, when choosing
a depletion strategy, LD, CS or D Columns
are recommended for certain applications to
obtain highest purities of the cell depleted
fractions. Both, depletion as well as positive
selection can also be performed by using
the respective autoMACS programs. After
magnetic separation, fluorescent staining of
the biotinylated material can be performed
by using streptavidin-fluorochromes.
Anti-Biotin MicroBeads # 130-090-485
# 130-048-701
# 130-058-701
# 130-048-801
# 130-090-485
# 130-048-101
# 130-048-602
# 130-047-501
# 130-048-401
# 130-047-101
# 130-047-201
# 130-047-301
# 130-048-501
# 130-047-401
 NEW PRODUCTS

Novel markers for human

blood dendritic cells
Recently Miltenyi Biotec‘s researchers
succeeded in the generation of new
monoclonal antibodies that are directed
against three presumably novel antigens
expressed on blood dendritic cells (BDC):
BDCA-2, BDCA-3 and BDCA-4 (1).
Antibodies against BDCA-2, BDCA-3,
BDCA-4 and CD1c (BDCA-1) allow the direct
detection and isolation of three distinct BDC
populations from human peripheral blood. Two
of the three BDC populations have been
well described in the literature (2-6), one is
a phenotypically plasmacytoid (lymphoid)
and the other a phenotypically myeloid BDC
population. Based on the expression of BDCA-
3, a third, also myeloid BDC population can
now clearly be distinguished from the other
two populations.
BDCA-2+BDCA-4+
plasmacytoid/lymphoid BDC
In fresh blood, BDCA-2 and BDCA-4 are
excellent markers for the plasmacytoid/
lymphoid BDC population which is CD123bright Kit, BDCA-2+BDCA-4+ BDC are directly
and CD11c- (Fig. 1). This population is also magnetically labeled with Anti-BDCA-4
known as NIPC (natural IFN-α producing MicroBeads.
cells). BDCA-2 and BDCA-4 are absent
from all other blood cells, including the two
other blood dendritic cell populations, CD34+
hematopoietic progenitor cells, T cells, B cells,
NK cells, monocytes, granulocytes, erythrocytes
and platelets (Table 1). Unlike BDCA-2,
BDCA-4 is also found on CD1a+ dendritic cells
that can be generated ex vivo from monocytes
and hematopoietic precursor cells (see product
information below). Anti-BDCA-2 and Anti-
BDCA-4 antibodies are both available now
as PE conjugates, Anti-BDCA-2 antibodies
Figure 1:
in addition as FITC conjugates. Two kits are BDCA-2+BDCA-4+ blood dendritic cells, May-
available now for direct magnetic isolation of Grünwald Giemsa stained.
BDCA-2+BDCA-4+ BDC from fresh blood or
lymphoid tissue. By using the BDCA-2 Cell
Isolation Kit, BDCA-2+BDCA-4+ BDC are
indirectly magnetically labeled with Anti-
BDCA-2-Biotin and Anti-Biotin-MicroBeads
following a procedure with no wash between
the first and the second incubation step.
When using the BDCA-4 Cell Isolation

Subset 1 Subset 2 Subset 3

(lymphoid) (myeloid) (myeloid)
Figure 2:
CD1c+ blood dendritic cells, May-Grünwald
BDC subset CD1c (BDCA-1)- CD1c (BDCA-1)+ CD1c (BDCA-1)- Giemsa stained.
specific antigens BDCA-2+ BDCA-2- BDCA-2-
BDCA-3- BDCA-3- BDCA-3+
BDCA-4+ BDCA-4- BDCA-4

markers also CD11c- CD11cbright CD11cdim

expressed on CD123bright CD123dim CD123-
other cell types
CD13- CD13+ CD13+
CD45RO- CD45RO+ CD45RO+
CD45RA+ CD45RA- CD45RA-
CD33+/- CD33++ CD33+
Figure 3:
% of PBMC (approx.) 0.41 ± 0.17 0.62 ± 0.01 0.04 ± 0.01 BDCA-3+ blood dendritic cells, May-Grünwald
Giemsa stained.

Table 1: Phenotypic characteristics of three distinct BDC subsets

Vol. 4 No. 2 2000 3

 NEW PRODUCTS

Plasmacytoid BDC isolated using

CD1c(BDCA-1)+ myeloid BDC PBMC the BDCA-4 Cell Isolation Kit

CD1c (BDCA-1)+ blood dendritic cells

(Fig 2) are CD11cbrightCD123dim. They

CD123

CD123
express none of the novel BDC antigens
BDCA-2, BDCA-3 and BDCA-4 (Table
1). Apart from the subset of BDC, CD1c
(BDCA-1) is also expressed on B cells.
Therefore, the CD1c (BDCA-1)+ Dendritic
Cell Isolation Kit which is now available Anti-BDCA-2 Anti-BDCA-2
uses CD19 MicroBeads for the depletion of
B cells before enriching CD1c (BDCA-1)+ CD1c+ BDC isolated using the
PBMC CD1c (BDCA-1) Dendritic Cell Isolation Kit
BDC based on indirect magnetic labeling
with Anti-CD1c-Biotin and Anti-Biotin
MicroBeads. For detection, CD1c (Anti-
BDCA-1)-FITC and CD1c (Anti-BDCA-1)- CD19

CD19
PE antibody conjugates are now available.

BDCA-3+ myeloid BDC CD1c (Anti-BDCA-1) CD1c (Anti-BDCA-1)

In fresh non-cultured blood, BDCA-3 is BDCA-3+ BDC isolated using the

PBMC
expressed on CD123-CD11cdim BDC (Fig. BDCA-3 Cell Isolation Kit
3). Like BDCA-2 and BDCA-4, BDCA-
3 is absent from CD34+ hematopoietic

Anti-BDCA-3
Anti-BDCA-3

progenitor cells, T cells, B cells, NK cells,

granulocytes, erythrocytes and platelets.
BDCA-3 is expressed at an extremely
low level on monocytes, CD1c+ BDC and
BDCA-2+BDCA-4+ BDC. Unlike CD1c+
BDC, BDCA-3+ BDC lack expression of
Fc receptors like CD32, CD64 and FcεRI.
Anti-BDCA-3 antibodies are available
now as FITC and PE conjugates. BDCA-3+
BDC can now be easily isolated based on
direct magnetic labeling with Anti-BDCA-
3 MicroBeads by using the BDCA-3 Cell
Isolation Kit.
CD14 CD14
Figure 4:
MACS Isolation of plasmacytoid BDC (upper panel of dot plots), CD1c+ myeloid BDC (middle
panel of dot plots) and BDCA-3+ myeloid BDC (lower panel of dot plots). The dot plots show
two-color stainings with Anti-BDCA-2 vs CD123, CD19. vs CD1c and Anti-BDCA-3 vs. CD14
before and after isolation of the respective BDC population (left and right panel of dot plots).
References:
1. Dzionek et al. (2000) J. Immunol. December 1 4. Olweus et al. (1997) Proc. Natl. Acad. Sci. U. S.
issue, in press A. 94:12551-12556
2. O‘Doherty et al (1994) Immunology 82:487-493 5. Kohrgruber et al. (1999) J. Immunol. 163:3250-
3. Robinson et al. (1999) Eur. J. Immunol. 29:2769- 3259
2778 6. Rissoan et al. (1999) Science 283:1183-1186

MACS Product information: BDCA-3 Cell Isolation Kit # 130-090-512

CD1c (BDCA-1) Dendritic Cell Isolation Kit # 130-090-506 Anti-BDCA-3-FITC # 130-090-513
CD1c (Anti-BDCA-1)-FITC # 130-090-507 Anit-BDCA-3-PE # 130-090-514
CD1c (Anti-BDCA-1)-PE # 130-090-508
Related MACS Products:
Blood Dendritic Cell Isolation Kit # 130-046-801
BDCA-4 Cell Isolation Kit # 130-090-532 CD1a MicroBeads # 130-051-001
Anti-BDCA-4-PE # 130-090-533 CD14 MicroBeads # 130-050-201
BDCA-2 Cell Isolation Kit # 130-090-509 Monocyte Isolation Kit # 130-053-301
Anti-BDCA-2-FITC # 130-090-510 CD34 Progenitor Cell Isolation Kit # 130-046-701
Anti-BDCA-2-PE # 130-090-511

4 © 2000 Miltenyi Biotec

 MACS PRODUCTS

Cytokine Secretion Assays - detection

and isolation of antigen-specific T cells
Antigen-specific T cells mediate specific 1
immune responses against many different Figure 1: Examples for applications
antigens and are of high interest for researchers The principle.
and clinicians in the fields of infectious Following antigen- for research on infectious disease, tumor,
diseases, tumor immunology, autoimmunity specific stimulation autoimmunity, allergy and for basic research
(1), MACS Cytokine
and allergy. But due to the usually low on the mechanism of cytokine secretion.
Catch Reagent is
frequencies and the difficult identification
attached to the cell
of these cells, research on antigen-specific Virus
surface (2) and 2
T cells is still, until today, an experimental binds secreted IL-4,
CMV-specific T cells could be detected and
challenge. The analysis of antigen-specific T IL-10 or IFN-γ (3). enriched according to secretion of IFN-γ,
cells is a prerequisite for the determination Bound cytokine IL-4 and IL-10. In addition to relatively high
of functional antigens in disease, for T cell is then stained frequencies of IFN-γ secreting T cells and
receptor (TCR) epitope mapping or for the with Cytokine lower frequencies of IL-4 secreting T cells, a
analysis of the TCR repertoire. Detection Antibody population of IL-10 secreting, CMV-specific
The recent development of the Cytokine for analysis and T cells could be detected (see Figure 2.). The
3
Secretion Assays now enables the sensitive (optional) with Cytokine Secretion Assays were also used for
detection and analysis of functionally active MACS MicroBeads studies on other viral infections such as HIV,
antigen-specific T cells. In contrast to other for enrichment (4). as presented by Douek (4).
methods, the cells remain viable and can be
stained with other markers in parallel. Apart Tumor
from the detection and analysis, the Cytokine Ölke and coworkers used the IFN-γ Secretion
Secretion Assays also allow to isolate viable Assay to isolate CD8+ Melan-A-specific T cells
4
antigen-specific T cells for research on (2). The cells were active in cytotoxicity assays
immunotherapy against cancer, infections or after further cultivation and the method thus
autoimmune diseases, for example. showed to be an efficient tool for generating
large numbers of peptide-specific T cells in
vitro that may be used for future adoptive T
The Cytokine Secretion Assays are now cell transfer in tumor immunotherapy.
Cytokine Secretion Assays Autoimmunity
for IFN-γ, IL-4 and IL-10 A Sample stimulated with CMV protein
before enrichment after enrichment
available for IFN-γ, IL-4 and IL-10 in two Figure 2. Example for the
different kit configurations: The Cytokine isolation of IL-10 secreting
Secretion Assay – Detection Kits are used for T cells from normal human
CD4-FITC

CD4-FITC

flow cytometric analyses of antigen-specific T PBMC. The cells were

cells like for IFN-γ-specreting virus-specific
T cells. With the Cytokine Secretion Assay
– Cell Enrichment and Detection Kits, the
secreting cells can additionally be isolated by
using Anti-PE MicroBeads. The enrichment
greatly increases the sensitivity of flow
cytometric detection. This is a prerequisite
for studies on rare antigen-specific T cells
with frequencies down to 1 in 106 such as
Cytomegalovirus (CMV)-specific T cells
incubated overnight with or
without CMV lysate. The IL-
10 Secretion Asssay was then
performed on both samples.
Anti-IL-10-PE Anti-IL-10-PE 432 IL-10+CD4+ cells were
isolated out of 107 total CMV
B Unstimulated control sample stimulated CD4+ cells. From
before enrichment after enrichment 107 total CD4+ cells of the
unstimulated control sample,
only 8 IL-10+CD4+ cells were
isolated. These results allow
CD4-FITC

CD4-FITC

secreting IL-10 (see below and Figure 2). The to calculate about 35,000 IL-
isolation of viable antigen-specific T cells also 10+ CMV-specific Th cells per
allows to perform subsequent experiments liter of blood for this donor.
like cell culture or functional assays (1-3).
Cytokine Secretion Assays have been used
Anti-IL-10-PE Anti-IL-10-PE

Vol. 4 No. 2 2000 5

 MACS PRODUCTS
Test Yourself
How much do you know about the Cytokine Secretion Assays? Find the answers on page 15.
1 4
What is the approximate detection
sensitivity of the Cytokine
Secretion Assays?
1 cell in 1,000 cells
1 cell in 10,000 cells
1 cell in 100,000 cells
1 cell in 1,000,000 cells
2
What starting material can be
used in combination with the
Cytokine Secretion Assays?
whole blood
Cytokine Secretion Assays (continued)
Alistar Gracie used Cytokine Secretion Assays
to study immunopathogenesis of rheumatoid
arthritis (7). He identified and isolated viable
Th1 cells from synovial membrane or fluid,
which now enables the ex vivo investigation of
Th1 cell regulation in purified cultures.
Allergy
Akdis and coworkers demonstrate that the
Cytokine Secretion Assays can be efficiently
used in purification and characterization of
allergen-specific cells (see report on page 9).
Basic research
Ouyang, Löhning and coworkers used the
Mouse IL-4 Secretion Assay to elucidate Th2
development (3 and MACS paper highlight,
page 14). The Mouse IL-4 Secretion Assay
was also used by Jane Hu-Li to investigate
the mechanism of IL-4 expression (5).
NK cells and monocytes
The Cytokine Secretion Assays do not only
allow research on antigen-specific T cells, but
also on other cytokine secreting cells.
IFN-γ secreting NK cells were isolated ex vivo
to study anti-allergic properties of NK cells in
For human cells
IFN-γ Secretion Assay
– Cell Enrichment and Detection Kit
– Detection Kit (PE*)
– Detection Kit (FITC)
IL-4 Secretion Assay
– Cell Enrichment and Detection Kit
– Detection Kit (PE*)
IL-10 Secretion Assay
– Cell Enrichment and Detection Kit
– Detection Kit (PE*)
6 © 2000 Miltenyi Biotec
PBMC What should always be considered
Spleen cells for most sensitive flow cytometric
Cultured cells analysis of antigen-specific T cells?
3
Always use histograms
What can be used for stimulation Exclude dead cells and monocytes
of cytokine secretion? Never work with Propidium Iodide
Peptides, Proteins staining
Supernatants/lysates
Whole cells
Staphylococcal Enterotoxin B
PMA/Ionomycin
polyallergic atopic dermatitis as recently
CMV-Tetramer-PE
reported by Cezmi Akdis (6).
John Campbell from Oxford identified
immature monocytes which spontaneously
produce IL-10 in peripheral blood stem cell
grafts (8). Using the IL-10 Secretion Assay
they were, for the first time, able to isolate and
purify these cells for use in functional assays.
A new protocol for the detection of cytokine Anti-IFN-γ-FITC
Figure 3. Detection of IFN-γ secreting, CMV-specific T
New developments
cells. PBMC of a CMV+ donor were first stained with
PE-labeled CMV tetramer (courtesy of D. Busch, TU
secreting cells directly from human whole Munich, Germany). The cells were then stimulated
blood is now available. This greatly reduces with CMV peptide for 2 h in vitro and afterwards
sample processing time by eliminating the stained for IFN-γ secretion with the IFN-γ Secretion
PBMC preparation and minimizes the Assay - Detection Kit (FITC).
quantity of blood required (0.25 ml blood References:
per test). * 1. Brosterhus (1999) Eur. J. Immunol. 29: 4053-4059. [573]
Apart from the IL-10 Secretion Assays for * 2. Oelke et al. (2000) Clin. Cancer Res. 6: 1997-2005. [663]
* 3. Ouyang et al. (2000) Immunity 12: 27-37. [597]
human cells, the development of the new Abstracts Cytokine Secretion Assay workshops 2000
IFN-γ Secretion Assay – Detection Kit (FITC) * 4. Douek D, AAI, Seattle, USA
now allows two color cytokine detection, for * 5. Hu-Li J, Löhning M, Radbruch A, Paul WE,AAI, Seattle, USA
* 6.Akdis M, Deniz G, Blaser K and Akdis CA,AAI, Seattle, USA
example of IFN-γ and IL-10 secreting cells. It * 7. Gracie A, London, UK
also enables counterstaining of cells stained * 8. John Campbell, London, UK
with peptide MHC-tetramers conjugated to References marked with * are MACS references or on file
Phycoerythrin (see Figure 3).
For murine cells
Mouse IFN-γ Secretion Assay
# 130-054-201 – Cell Enrichment and Detection Kit # 130-090-517
# 130-054-202 – Detection Kit (PE*) # 130-090-516
# 130-090-433 Mouse IL-4 Secretion Assay
– Cell Enrichment and Detection Kit # 130-090-515
# 130-054-101 – Detection Kit (PE*) # 130-090-479
# 130-054-102
# 130-090-435
# 130-090-434
 CUSTOMER REPORT

Research on Malaria is supported by MACS: The high gradient magnetic field generated in a MACS
Column enabled Anne-Catrin Uhlemann and Trine Staalsoe to easily enrich Plasmodium-infected
erythrocytes which become paramagenetic at a late stage of infection.

Analysis of Plasmodium
falciparum-infected
red blood cells
Anne-Catrin Uhlemann*a, Trine Staalsoe*b, Mo-Quen Klinkert a, and Lars Hviid b
a Department of Human Parasitology, Institute for Tropical Medicine, University of Tuebingen, Wilhelmstrasse 27,
72074 Tuebingen, Germany, b Center for Medical Parasitology, Department of Infectious Diseases, Rigshospitalet,
University of Copenhagen, Denmark
* These authors contributed equally to this work.

Introduction

All symptoms of Plasmodium falciparum
malaria are associated with the multiplication
of asexual stage parasites in the red blood
cells (RBC) of the patient.
Within RBC, the parasites mature from ring
forms through trophozoites, before dividing
to form schizonts, each containing up to 16
daughter parasites ready to infect new RBC.
This process repeats itself every 48 hours.
Techniques have been developed to maintain
the asexual parasite life cycle continuously
in vitro, and fairly high parasitaemias can
be achieved when using culture-adapted
parasites. However, this is often difficult
with primary isolates obtained from
patients, and various density-gradient or
osmotic fragility techniques have been
developed to synchronize cultures and
increase parasitaemia. Unfortunately, these
techniques are generally laborious.
Malaria parasites digest RBC haemoglobin,
leaving insoluble high-spin oxidized
haem products, which polymerize into a
pigmented substance, called haemozoin. The
paramagnetic properties of haem products,
in contrast to the diamagnetic low-spin
oxyhaemoglobin, led Paul et al. (1) to propose
a high-gradient magnetic separation method
as early as 1981. Here, we describe a refined
technique using a variety of MACS systems
to synchronize and enrich in vitro cultures of
P. falciparum.
Materials and Methods
Parasite culture and preparation of
infected RBC
Asexual blood stage parasites were
cultured in human type 0 erythrocytes by
a modification (2) of the original method
developed by Trager and Jensen (3). Several
long-term isolates, such as Binh and 3D7, as
well as primary patient isolates, were used
in this study. Infected RBC (IRBC) cultures
were washed once in PBS and resuspended
in PBS supplemented with 2% bovine serum
albumin (BSA). All reagents were purchased
from Sigma (Deisenhofen, Germany) unless
otherwise stated.
Magnetic separation
Different Separation Units (MidiMACS,
VarioMACS and SuperMACS) and
Columns (LD and CS) were prepared and
used according to standard procedures. For
CS Columns, a 20 G flow resistor was applied.
The flow velocity was further reduced
by turning the stopcock. Parasite culture
material in PBS/2% BSA was applied to the
column, which was subsequently washed at
least three times with PBS/2% BSA (LD:
approx. 1 ml; CS: approx. 15 ml). When no
more RBC from the parasite culture were
apparent in the flow-through, the magnet was
removed, and the cells retained in the column
were eluted in PBS/2% BSA.
Assessment of yield and purity
To determine the purification efficiency,
aliquots of the original culture, flow-through
and eluate were spread on microscope slides,
fixed in methanol, stained with Giemsa,
and analyzed under the microscope. In
addition, samples were analyzed by flow
cytometry. For this procedure, RBC were
stained with ethidium bromide (1 μg/1x105
RBC), allowing DNA-containing IRBC
to be distinguished from uninfected RBC.
Subsequently, samples were incubated for 30
min at room temperature and washed 3 times
prior to analysis by flow cytometry. Data
was analyzed by WinMDI software (http:
//facs.scripps. edu/software.html).
Downstream applications after MACS
separation
Eluted late-stage parasites were put back
into culture after adjusting the haematocrit
to about 5% and the parasitaemia to

Vol. 4 No. 2 2000 7

 CUSTOMER REPORT
0.5–5%. After one day, re-invasion of new
RBC was assessed by GIEMSA staining.
For preparation of the invasive merozoite
parasites, undiluted MACS eluates
containing late-stage-enriched parasites were
taken back to culture until free merozoites
were released into the culture medium. The
depleted flow-through fraction, containing
mainly ring-stage parasites, was either used as
such for further culture, as described above,
or subjected to an additional synchronization
step via sorbitol treatment.
A
Events
1.8 %
B P. falciparum-infected RBC from routine
Events
0.3 %
C contain mainly ring forms and some early
Events
84.9 %
ETHIDIUM BROMIDE
Figure 2.
Flow cytometric analysis of ethidium bromide-labeled
erythrocytes. A: Before purification. B: MACS flow-
through containing early-stage (ring-form) parasites.
C: MACS eluate containing concentrated late-
stage-infected erythrocytes. Percentages indicate
the proportion of ethidium bromide labeled (i.e.,
parasite-containing) erythrocytes.
8 © 2000 Miltenyi Biotec
(A)
Figure 1.
Giemsa-stained thin smears of parasitized culture material obtained before (left) and after enrichment of late-
stage-infected erythrocytes by MACS. Note low parasitaemia and presence of both early-stage (ring-form, blue
arrow) and late-stage parasites (red arrow) before purification (A), and the high parasitaemia of erythrocytes
infected by haemozoin-containing (brownish granules) late-stage parasites in the MACS eluate (B).
Results and Discussion
In this study we have used different MACS
Separation Units and Columns to separate
blood-stage parasite cultures. Purity and
efficacy of the procedure were assessed by
blood smears (Fig. 1) and flow cytometric
analysis (Fig. 2). By using CS Columns
and VarioMACS, parasitaemias of <3%
(20 ml culture, 5% haematocrit) could be
increased to 70-90%, depending on the stage
of the trophozoites as well as the age of the
RBC. Further enrichment of up to 95% was
achieved when the eluate was passed through
a second column.
The flow-through sample was found to
trophozoites. Both re-application to a second
column and combining MACS separation
with sorbitol treatment yielded a highly
synchronized culture. Similar results were
obtained by using the SuperMACS.
Comparable results were obtained with the
LD Column in the MidiMACS, although
the LD Column, which is designed for cell
depletion, has a relatively slow flow rate.
Furthermore, only up to 250 μl RBC pellet
could be applied to the LD Column (approx.
2,5 x 109 RBC dissolved in 1 ml), compared
to 1 ml for the CS Column (approx. 10 10 RBC
dissolved in 10 ml). Nevertheless, the LD
Column has the advantage of being cheaper,
and is useful when only small volumes of
IRBC are required.
(B)
We have used IRBC concentrated by
MACS technology for several downstream
applications, such as: i) direct usage of enriched
late-stage parasites in flow cytometry assays
(2), ii) high-yield harvest of free merozoites
4 to 8 hours after MACS separation, and
iii) efficient culture synchronization, using
either the late-stage-enriched parasites from
the MACS eluate or the purified ring-stage-
infected RBC from the flow-through.
In summary, exploitation of the paramagnetic
properties of haemozoin is a very powerful
technique in routine culture and analysis
of P. falciparum parasites. The method is
both easy to perform, rapid, and non-toxic,
allowing the enrichment or depletion of late-
stage-infected RBC without the need for any
labeling or processing of culture material.
References
1. Paul et al. (1981) Lancet 2:70-71
* 2. Staalsoe et al. (1999) Cytometry 35:329-336 [837]
3. Trager and Jensen (1976) Science 193:673-675
The reference marked with * is a MACS reference.
LD Columns # 130-042-901
CS Separation Columns # 130-041-305
MidiMACSStarting Kit # 130-042-301
VarioMACS Starting Kit # 130-043-102
SuperMACS II Starting Kit # 130-044-103
 CUSTOMER REPORT

New ways of allergy research are possible as life antigen-specific T cells can be isolated by using the
MACS Cytokine Secretion Assays. Here, Mübeccel and Cezmi Akdis and Kurt Blaser report on the
isolation and analyzation of birch pollen antigen-specific T cells from allergic patients.

Purification of allergen-specific
T cells from allergic patients
based on IL-4 and IFN-γ secretion
Mübeccel Akdis, Kurt Blaser, Cezmi A. Akdis
Swiss Institute of Allergy and Asthma Research
(SIAF), Davos, Switzerland
Correspondence address: Dr. Mübeccel Akdis
Swiss Institute of Allergy and Asthma Research
(SIAF), CH-7270 Davos, Switzerland
email:akdism@siaf.unizh.ch

Introduction

Development of specific immune responses
requires the encounter of antigens with the
cells of the immune system and interaction
between T and B lymphocytes and other
antigen presenting cells (APC) (1). The
presentation of soluble antigens to T cells by
APC involves a complex process including
Ag capture, processing and presentation
of Ag fragments by MHC molecules (1,2).
Ag presentation by different types of APC
can promote the development of distinct
cytokine-secreting T cell subsets (3). Th1
cytokine profiles, characterized by IL-2, IFN-
γ and TNF-β, are implicated in cell-mediated
defense against certain intracellular
microorganisms and in promotion of memory
IgG responses (4). Th2 cells secrete IL-4, IL-
5 and IL-13 and predominantly mediate IgE
responses and allergic inflammation (4). By
the aim of cellular and molecular parameters
related to particular immune reactions,
research interests have now focused on
differentiation of cytokine expression. A
recently developed technique enables the
isolation of antigen-stimulated IL-4 and
IFN-γ producing cells from peripheral blood
(5). Here we describe the purification of Bet
v 1-specific IL-4 and IFN-γ producing T cells
from peripheral blood of birch pollen allergic
patients.
Materials and Methods
PBMC from birch pollen-allergic patients
were isolated by Ficoll (Biochrom, Berlin,
Germany) density gradient centrifugation of
peripheral venous blood. Cells were washed
three times and resuspended in RPMI 1640
medium supplemented with 1 mM sodium
pyruvate, 1% MEM nonessential amino
acids and vitamins, 2 mM L-glutamine, 100
U/ml penicillin, 100 µg/ml streptomycin, 50
µM 2-ME (all from Life Technologies, Basel,
Switzerland) and 10% heat inactivated FCS
(Sera-Lab, Sussex, England) (6). 2.5x107 cells
were stimulated with 20 µg/ml rBet v 1 (gift
of Dr. Rudolf Valenta, Institute for General
and Experimental Pathology, Wien, Austria)
in 5 ml medium in 6 well plates (Costar
Corp., Cambridge, MA, USA) in duplicates.
Cells were incubated in humidified 5% CO2
for 5 to 10 h. After ex vivo stimulation, cells
were harvested and labeled for 10 min at
a concentration of 108 cells/ml in ice cold
medium with 50 µg/ml of Cytokine Catch
Reagent, i.e. anti-IFN-γ /CD45 or anti-IL-
4/CD45, Ab-Ab conjugates (Miltenyi Biotec,
Bergisch Gladbach, Germany) (5). Then the
cells were diluted with 37oC warm medium
to a final concentration of 106 cells/ml and
allowed to secrete for 45 min at 37oC. After
capturing secreted cytokines at their surface,
cells were centrifuged at 300xg for 5 min at
4oC and resuspended at a concentration
of 108 cells/ml in ice cold buffer containing
0.5% BSA and 5 mM EDTA (both from
Sigma Chem. Co. St. Louis, MA) in PBS.
The cells were then stained with 5 µg/ml
PE-conjugated anti-IFN-γ and anti-IL-4
for 10 min, at 4oC. The cells were washed
and resuspended in 400 µl of buffer and
magnetically labeled for 15 min at 4oC with
100 µl of anti-PE MicroBeads (Miltenyi
Biotec) (5). After washing, the cells were
applied onto an AS Column placed in a
VarioMACS. The cells were elevated 5 times
with 2 ml of buffer after separation of the
column from the magnet and rinsed after
adjusting the column to the magnet. This
intense rinsing procedure enabled a high
enrichment rate. Finally, the retained cells
were eluted after having been removed from
the magnet. The cell samples were counter-

Vol. 4 No. 2 2000 9

 CUSTOMER REPORT

stained by FITC labeled anti-CD4 mAb and
analyzed in a flow cytometer (Epics XL,
Coulter Corp. Hialeah, FL, USA).
Purified Bet v 1-specific IL-4 and IFN-γ
secreting cells (5x103-10x103) were stimulated
in complete culture medium with 25 U/ml IL-
2 and the following combination of mAbs to
T cell surface molecules: anti-CD2 (4B2 and
6G4, each 0.5 mg/ml), anti-CD3 (0.5 mg/ml),
and anti-CD28 (0.5 mg/ml) (7). Expanded
IL-4 and IFN-γ producing cells were washed
and restimulated with a combination of mAbs
to CD2/CD3/CD28 molecules as above (5 x
105 cells in 500 µl medium, in 48 well plate,
Costar Corp.) for three days in triplicates.
Supernatants were harvested and cytokines
were determined by solid phase sandwich
analyzed after restimulation with anti-CD2,
anti-CD3, anti-CD28 mAb combination. As
shown in Fig. 2, Bet v 1-specific IL-4 secreting
cells secreted higher IL-4, IL-5, IL-10 and IL-
13 than IFN-γ secreting cells. Whereas Bet v 1-
specific IFN-γ secreting cells secreted higher
amounts of IFN-γ. These results demonstrate
that cytokine secretion assays operate in
allergy models and can be efficiently used in
purification and characterization of allergen-
specific cells.
before separation
Figure 1:
PBMC from birch pollen
allergic individuals were
stimulated with rBet v 1 and
IL-4 and IFN-γ secreting
References
1. Lanzavecchia (1996) Curr. Opin. Immunol. 8: 348-354
2. Nelson et al. (1995) Nature 375: 145
3. Akdis CA et al. (1998) Eur. J. Immunol. 28: 914-925
4. Mosmann and Sad (1996) Immunol. Today 17: 138-146
* 5. Brosterhus et al. (1999) Eur. J. Immunol. 29: 4053-4059 [573]
6. Akdis CA, et al. (1996) J. Clin. Invest. 98: 1676-1683
* 7. Akdis M et al. (1999) J. Immunol. 163: 466-457 [862]
8. Akdis M et al. (1997) J. Immunol. 159: 4611-4619
9. Akdis CA et al. (1998) J. Clin. Invest. 102: 98-106
The references marked with * are MACS references.
after separation

CD4
CD4
ELISAs for IFN-γ, IL-4, IL-5, IL-10 and IL-13 cells were isolated by
MACS. Cells were stained
as previously described (6,8,9). with FITC conjugated anti-
CD4 and PE conjugated
anti-IL-4 or anti-IFN-γ.
Live lymphocytes were
Results and Discussion gated and analyzed by flow
IL-4 IL-4
cytometry.
CD4+ memory effector cells of allergic
patients secreted IL-4 and IFN-γ after 5 to 10
h of stimulation with the major birch pollen
allergen, Bet v 1 (Fig. 1). Compared to the
nonstimulated control samples, a significantly

CD4
CD4

higher proportion of IL-4 and IFN-γ secreting

cells were detectable after Bet v 1 stimulation.
These cells were isolated with a precursor
frequency of less than 1 in 5000. Interestingly,
spontaneous IL-4 secretion of CD4- T cells
IFN-γ IFN-γ
was observed in allergic individuals. After
the enrichment procedure, 59.7 % of the IL-
4 secreting cells were in the CD4+ subset, Bet v 1-specific IL-4 secreting cells Bet v 1-specific IFN-γ secreting cells
however 36.8% of a CD4- cells also secreted
IL-4. Unstimulated cells did not release any IFN-γ IL-4 IL-5 IL-10 IL-13
IFN-γ but secreted IL-4 spontaneously in 3 0.8 40 6 40
allergic individuals. Similarly, spontaneous
cytokine (ng/ml)

2.5 5
0.6 30 30
IL-4, IL-5 and IL-13 secreting cells were 2 4
demonstrated from atopic dermatitis patients
1.5 0.4 20 3 20
(7,8). Interestingly, Bet v 1 stimulated IFN-γ
1 2
secretion from PBMC of allergic individuals 0.2 10 10
and Bet v 1-specific IFN-γ secreting CD4+ cells 0.5 1
could be isolated with a purity of 97.7 %. 0 0 0 0 0
We further investigated the Th1 and Th2
cytokine pattern of Bet v 1-specific cells Figure 2.:
rBet v 1-specific IL-4 and IFN-γ secreting T cells were expanded with IL-2 and mAbs to CD2/CD3/CD28
in allergic individuals. Because of the low
molecules. After two weeks, cells were washed and restimulated with the same mAb combination. Cytokines
numbers of Bet v 1-specific cytokine-releasing were determined from 72 h supernatants by ELISA.
cells obtained after purification, T cells
had to be expanded in vitro. The cells were
IFN-γ Secretion Assay - Cell Enrichment and Detection Kit # 130-054-201
stimulated with a combination of anti-CD2,
IL-4 Secretion Assay - Cell Enrichment and Detection Kit # 130-054-101
anti-CD3, anti-CD28 mAbs in the presence
MS Column # 130-042-201
of IL-2 for optimal T cell proliferation. Th1
MiniMACS Starting Kit # 130-090-312
and Th2 cytokine profiles of the cells were

10 © 2000 Miltenyi Biotec

 MEETING REPORT

CD133 (AC133) - from basic

research to clinical relevance
Workshop during the 29th Annual Scientific Meeting of the International
Society for Experimental Hematology, July 8, 2000, Tampa, USA

Markus Bernards and Sandra Werres,

Milteny Biotec GmbH, Friedrich-Ebert-Str. 68, 51429 Bergisch Gladbach, Germany

The results presented by four researchers

Number of CFC per 1000 cells

500 Lin- CD34-CD38-

during the last AC133 workshop revealed the In addition, Bhatia and coworkers could show
400
significance of AC133 (recently designated that the AC133+CD7- subpopulation did not
as CD133) as a marker not only for CD34 300
only exist in cord blood but also in adult
negative hematopoietic stem cells but also mobilized peripheral blood, surprisingly in a
for other developmentally early cells. The 200 much higher percentage (4 % of the CD34-
5-transmembrane antigen seems to be also CD38-Lin- population). As seen in cord
100
expressed on endothelial precursor cells as blood, these cells were able to differentiate
well as probably on other developmentally 0 into AC133+CD34+ cells after culturing them
early cells such as muscle and neural CD7- CD7+ AC133- AC133+ for 10 days (Fig. 2). The number of CD34+
progenitor cells. Further, the relevance of in the CD34+CD38- fraction decreased
Plating Efficiency:
AC133 selection for future clinical tumor cell 1 in 20 1 in 140 1 in 100 1 in 2.6 distinctly in culture due to differentiation
purging was shown. processes.
Figure 1:
The subfraction of AC133+CD7- cells within At the end of his talk, Bhatia gave an outlook
Lin-CD34-CD38- population contains primitive
on stem cell plasticity. His preliminary data
progenitors detectable in vitro.
AC133 in CD34- populations may indicate that AC133 is rather a marker
specific for stem cells with omnipotential
Interested in CD34- cells that were shown to found only in a small, dim cluster. „The capacity than for cells restricted to the
have reconstitutive hematopoietic potential, surprising result“, Bhatia pointed out, „was hematopoietic lineage.
Mickie Bhatia from the University of the fact, that out of the limited amount of
Western Ontario in London, Canada and colony forming capacity the entire CD34-
coworkers described primitive hematopoietic fraction has, it seemed to be preferentially
progenitor AC133+CD7- cells within a Lin- found in the AC133+ fraction“ (Fig. 1). The
CD34-CD38- population. After a lineage number of cells that gave rise to colonies
depletion of mononuclear cord blood cells was equivalent to that of the CD34+ cells.
they got CD34+ cells as well as a small CD34- When injected into NOC/SCID mice, as few
population, which had a comparatively low as 30 AC133+CD7- cells from the Lin-CD34-
CD38

colony forming capacity. However, when
injected into irradiated NOD/SCID mice,
the hematopoietic system of a subset of these
mice was reconstituted. The ability to engraft
the mouse‘s bone marrrow was restricted to
CD38- cells of the Lin-CD34- population.
Further characterisations showed that the
majority of Lin-CD34-CD38- cells expressed
CD7 but not AC133. AC133+ cells were
CD38- population allowed the engraftment
of the bone marrow after six weeks as could
be shown by Southern Blot analysis. In
contrast, 44,000 AC133- cells did not give
any signal in a parallel experiment. After
four days of cell culturing the AC133+ and CD34
CD7- subpopulations showed to some extent
the ability to differentiate into CD34+ cells.
Figure 2:
This was not the case in the AC133- and CD7+ Differentiative capacity of AC133+CD34- subset
populations of the CD34-CD38-Lin- cells. after 10 days ex vivo culture.

Vol. 4 No. 2 2000 11

 MEETING REPORT
AC133 on endothelial
stem cells
Evidence for the expression of AC133 on
circulating human CD34+ cells that identify
a population of endothelial precursors
was presented by Shahin Rafii from the
Cornell University Medical College in New
York, USA. Rafii and coworkers isolated
AC133+VEGFR-2+CD34+ cells from various
sources such as fetal liver, peripheral blood,
mobilized peripheral blood and cord blood.
In vitro, these non-adherent cells gave rise to
an endothelial monolayer after culturing on
an extracellular collagen matrix together with
growth factors. When the AC133+VEGFR-2+
cells were injected in lethally irradiated NOD/
SCID mice they contributed to angiogenesis
into an implanted human leukemic tumor
after a chimeric bone marrow had been
reconstituted. When injected into knock-out
mice that are unable to grow tumors due to
the lack of three Id alleles AC133+VEGFR-
2+ cells were able to reconstitute the tumor
growing capacity. “In terms of vascular
healing we believe that these bone marrow-
derived cells are mobilized in response to
VEGF which can be derived by platelets
and contribute to vascular healing.” Rafii
pointed out. “And if this is true, harvesting
of these cells provides us with a powerful
tool to study them and to target the vascular
system for gene therapy and also for different
pathological processes.”
AC133 in cord blood
Stefano Papa from Urbino, Italy talked about
his and his coworkers‘ investigations on the
expression of AC133 positive hematopoietic
progenitor cells with relation to the
expression of various other markers. In CD34
and AC133 subsets the definition of positive
and negative cells is difficult for antigens
such as CD38. Therefore they standardized
the samples by setting the median for each
channel of the flow cytometer and by taking
lymphocytes as an internal control. Being
interested in the question whether AC133
cells undergo differentiation through a higher
expression of the antigen they analyzed 25
umbilical cord blood samples phenotypically.
“We found a lot of differences from cord
blood to leukapheresis product. The only
12
A
were found only in the AC133bright and the
CD34bright population“ Papa pointed out
(Figure 3 A). In addition they could show
the existence of an AC133dim population
AC133-PE
(figure 3 B). CD90 was also present on a
subset of the AC133+ cells, but most of the
latter were CD34+CD90-. In 5 of the 25 cord
blood samples they discovered a CD34dim as
well as a CD34superbright subpopulation within
the CD90+ population (Figure 4). The same
was true for CD135 where also a superbright
CD34+ population could be seen in addition
CD38-FITC
to a CD34dim subpopulation. In just one of
the cord blood samples they discovered an
B CD90+AC133superbright population. Future
experiments will show whether the CD90
positive cells are likely to be commited to
be stem cells.
Transformed SSC
Ulrike Köhl from the University Hospital
Frankfurt, Germany reported on the first
AC133 for tumor cell
depletion
AC133-PE results of clinical scale purification by AC133
selection. She compared AC133 selection
results of freshly isolated (n=2) as well as
Figure 3: cryopreserved (n=4) peripheral blood stem
In both, cord blood samples as well as apheresis
cells (PBSC) from Neuroblastoma patients
product CD38- cells were only found in the
AC133bright population (A, green). In addition, and PBSC from healthy donors (n=5) with
a AC133dim population could be shown in cord CD34 selected transplants (n=75). Aim
blood (B). of the study was to observe the selection
efficacy in terms of CD34 purity and yield as
well as monitoring of tumor cell depletion
in patient samples and T cell depletion
in donor samples in comparison to CD34
based selection. The selection strategy
T-cell depletion
CD34 selection 3.9 - 5.6 log
CD34-PC5
AC133 selection 4.5 - 4.8 log
10 11
10 10
109
108
107
106
105
CD90-FITC 104
thing that is equal for both is that CD38- cells 103
before selection after selection
Figure 4: Figure 5:
In 5 of 25 cord blood samples a CD90+ AC133 selection of apharesis products from pediatric
CD34superbright population was found in addition to patients with starting frequencies higher than 1%.
a CD90+CD34dim population.
© 2000 Miltenyi Biotec
 MEETING REPORT

included labelling of progenitor cells with [%] 100

either AC133/1-antibody or CD34-antibody
(QBEND10) conjugated to microbeads and 80
subsequent positive selection employing the
SuperMACS or CliniMACS device. 60
AC133 selection of apheresis products
from pediatric patients with CD34 starting 40 Figure 6:
Detection of
frequencies higher than 1 % resulted in tumor cells
20
consistently high CD34 purities (≥ 98 %) in samples of
comparable to CD34 selection results (Fig. 7). Neuroblastoma
0 patients.
Lower CD34+ frequencies in the apheresis bone marrowleukapheresis stem cells stem cells cell lines peripheral
product as observed with 5 healthy donors product after AC133 after CD34 Neuroblastomablood
selection selection lymphocytes
resulted in apparently lower CD34 purities
after AC133 selection which has also been
shown in some cases for CD34 purification.

Tumor cell contamination in Neuroblastoma 100

patients was found in more than 60 % of bone
CD34+ after selection [%]

marrow samples prior to transplantation and 90

in about 44 % of leukapheresis products as 80
detected by RT-PCR with 2 different markers
and immunhistochemistry with 3 different 70
markers. Upon AC133 or CD34 selection Figure 7:
60
no residual neuroblastoma cells could be Effect of CD34+
AC133 selection, patients content in
detected in the transplant anymore (Fig. 6). 50
AC133 selection, healthy donors apheresis product
CD34 selection on the purity after
It is essential for allogeneic transplantation AC133 selection.
approaches that consistently high T cell 0 1 2 3 4 5 6 7 8 9
depletion could be obtained by AC133 cell
CD34+ ln leukapheresis product [%]
selection (4.2-4.8 log). The depletion efficacy
was comparable to that obtained with CD34
selection (3.9-5.6 log, Fig. 5).
in cases of CD34 positive tumors and may MACS product information
Köhl concluded that these first results of help to improve the clinical outcome in these
AC133 Cell Isolation Kit # 130-050-801
large scale positive AC133 selection show patients.
CliniMACS AC133 Reagent* # 172-01
a high purity of progenitor cells with high
Anti-AC133/1-PE** # 130-080-801
tumor and T cell depletion comparable
Anti-AC133/1 pure # 130-090-422
to CD34 selection (Table 1). In the future
Anti-AC133/2-PE** # 130-080-901
AC133 purification may be an alternative
Anti-AC133/2 pure # 130-090-423
to CD34 selection for tumor cell purging

Purity CD34+ selection (patients) CD34 [%] 97.1 ± 2.3 (93.8 – 99.4) n = 40
CD34+ selection (donors) CD34 [%] 96.5 ± 2.8 (93.8 – 99.1) n = 35
AC133+ selection (patients) CD34 [%] 98.1 (98.0 – 98.1) n=2
AC133+ selection (donors) CD34 [%] 88.4 ± 6.3 (79.5 – 95.2) n=5

Recovery CD34+-selection CD34 [%] 64.7 ± 14 (41.8 – 87.0)

AC133+-selection CD34 [%] 60.0 ± 22 (22.0 – 94.1)

Depletion T cells: by CD34+ selection 3.9-5.6 log10

by AC133+ selection 4.2-4.8 log10
Tumor cells: no residual Neuroblastoma cells detectable after selection

Table 1: Summary of selection results reported by Köhl.

* In the U.S., the CliniMACS Cell Selection System is limited by FDA regulations to use under the Investigational Device Exemption, or related provisions, of Federal Law.
** Phycoerythrin, U.S. Patent 4,520,110; European patent 76,695; Australian Patent 548,440; Canadian Patent 1,179,942; Japanese Patent 1,594,827.

Vol. 4 No. 2 2000 13

 MACS NEWS

MACS Paper
Highlights
Stat6 independent Gata 3

Autoactivation directs IL-4-independent
Th2 development and commitment.
Ouyang W; Löhning M; Gao Z;
Assenmacher M; Ranganath S; Radbruch
A; Murphy KM. Immunity 12: 27-37. 2000
[597]
Ouyang, Löhning and coworkers used the
Mouse IL-4 Secretion Assay to study Th2
development. They could show that IL-4
secreting Stat6-deficient T cells stably expressed
GATA-3 and a Th2 phenotype. GATA-3 exerts
Stat6-independent autoactivation that provides
an IL-4 independent molecular basis for
stabilized Th2 commitment. Additionally, the
results show that conventional T cells may be
capable of providing an early IL-4 independent
source of IL-4.
Spleen cells from D011.10 TCR-transgenic
Stat6-deficient mice were stimulated with
specific ovalbumin peptide (OVA) and
antigen presenting cells or with PMA/
Ionomycin. The cells were then analyzed for
IL-4 secretion by using the IL-4 Secretion
Assay or by intracellular cytokine staining.
IL-4 secreting cells labeled with the
Cytokine Secretion Assay were isolated with
LS columns and MidiMACS. The enriched
IL-4 secreting cells as well as the depleted
non-IL-4 producing cells were subsequently
expanded. The authors could show that there
is no cross-contamination of the secreted IL-
4 to non-IL-4-producing cells during the
Cytokine Secretion Assay. The detection
of IL-4 secreting cells with the Cytokine
Secretion Assay showed to be as sensitive
as with standard intracellular staining and
nearly all cytokine secreting cells stain also
for intracellular IL-4 and vice versa.
IL-4 Secretion Assay - Cell Enrichment
and Detection Kit (human) # 130-054-101

γ/δ T cells in early tumor defense

Human γ/δ T lymphocytes exert
natural and IL-2-induced cytotoxicity
to Neuroblastoma cells. (2000);
Schilbach, KE; Geiselhart, A; Wessels,
JT; Niethammer, D; Handgretinger, R; J.
Immunother. 23: 536-548. [866]
Schilbach and co-workers investigated the
role of γ/δ T cells in early tumor defense.The
authors could for the first time report about
natural and inducible cytotoxicity of γ/δ T
cells against human neuroblastoma cells.
Cytotoxic properties of γ/δ Τ cells together
with their easy propagation, purification and
missing alloreactivity indicate a potential of
γ/δ T cells in adoptive immunotherapy.
Human γ/δ T lymphocytes were isolated
from PMBC of healthy donors by using the
TCR γ/δ MicroBead Kit and LS Columns.
Alternatively, γ/δ T cells were magnetically
sorted after propagation by PBMC bulk
expansion. Positively selected γδ T cells
were either cultured or used immediately
after separation for cytotoxicity assays. The
negative fraction was used for generation of
autologous feeder cells or as negative control
in cytotoxicity assays. Mixed lymphocyte
culture was carried out to investigate
allogenic reactivity of γ/δ T cells.
TCR γ/δ MicroBead Kit # 130-050-701

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MEDICA 2000, Düsseldorf, Germany 22.- 25. November 2000
Herbsttagung der Deutschen Gesellschaft für Immunologie, Düsseldorf, Germany 29. November - 02. Dezember 2000
ASH, Annual Meeting of the American Society of Hematology, San Francisco, USA 01.- 05. Dezember 2000
3rd International Symposium on Minimal Residual Cancer, Hamburg, Germany 16.- 18. Februar 2001

14 © 2000 Miltenyi Biotec

 TEST YOURSELF

Test Yourself: The answers

1
What is the approximate detection Figure 1:
A Sample stimulated with PLA
sensitivity of the Cytokine Example for the highly
Secretion Assays? sensitive detection of
Phospholipase A (PLA)-
specific, IL-4 secreting
The detection sensitivity of the Cytokine
CD4+ T cells. PLA is an

CD4-FITC

CD4-FITC
Secretion Assay – Detection Kits is in the allergenic component of bee
range of 1 cell in 1,000 cells. It depends on the venom. PBMC from a bee-
detection limit of the flow cytometric analysis venom allergic donor were
incubated with PLA (A) or
and requires acquisition of appropriate cell without antigen (B). The
numbers, i. e. 50,000 - 200,000 cells. IL-4 Secretion Assay - Cell
After enrichment of the cytokine secreting Enrichment and Detection
Kit was performed on both Anti-IL-4-PE Anti-IL-4-PE
cells by using the the Cytokine Secretion samples, the IL-4 secreting
Assay – Cell Enrichment and Detection cells were enriched over two
Kits, the detection sensitivity increases successive MS Columns. 173 B Unstimulated control
IL-4+CD4+ cells could be
dramatically, up to 1 cytokine secreting cell isolated from 106 total CD4+
in 1,000,000 cells can then be detected. This cells after PLA stimulation.
requires the use of appropriate cell numbers The unstimulated control
sample shows 0 IL-4+CD4+

CD4-FITC

CD4-FITC
per test. cells. These results allow to
calculate a total number of
about 140,000 IL-4+ PLA-

2
What starting material can be specific Th cells per liter of
used in combination with the blood for this donor.
Cytokine Secretion Assays?
Anti-IL-4-PE Anti-IL-4-PE
For the detection and isolation of cytokine
secreting cells, it is recommended to start from
fresh (alternatively frozen) PBMC, single cell
preparations from spleen, lymph nodes or
4 What should always be considered for most sensitive
flow cytometric analysis of antigen-specific T cells?
other tissues or cell lines. In addition, for We recommend to always exclude monocytes and dead cells from the analysis.
flow cytometric analyses of human cytokine Monocytes can be excluded via scatter properties or, more reliably, by staining with CD14-
secreting cells, a whole blood protocol is now PerCP and exclusion in FL-3. Dead cells should be excluded in the FL-2 versus FL-3 plot after
available (see at www.miltenyibiotec.com: staining with Propidium Iodide.
MACS, Technical Support, Special Protocols).
Figure 2:

3 What can be used for stimulation

of cytokine secretion?
Example for the analysis of IFN-
γ secreting murine T cells. The
Mouse IFN-γ Secretion Assay
A B
CD45R, CD11b, PI

– Detection Kit was performed

Antigen-specific restimulation of the cells after stimulation of murine
can be performed with peptides, proteins, spleen cells with 10 µg/ml SEB
SSC

viral- and bacterial antigens or crude antigen
preparations. The antigen may directly be
added to PBMC. Alternatively antigen-
presenting cells pulsed with antigen or
transfected cells may be used for stimulation.
Staphylococcal Enterotoxin B (SEB) is an
ideal high control for the stimulation of
human PBMC. We recommend to stimulate
for 3 hours or overnight with 10 µg SEB
for 16 hours. Macrophages and B
cells were stained with CD11b-
PerCP and CD45R-PerCP,
respectively. T cells were stained
with CD90-FITC. Propidium
Iodide was added just prior to the
flow cytometric analysis at a final FSC IFN-γ
C
concentration of 0.5 µg/ml.
In a first gating step, debris
and some dead cells are gated
out via scatter properties (A).
Subsequently, macrophages,
CD90

per ml. In general, cells should be cultured
at 107 cells/ml and 5x106 cells/cm2 for
optimal stimulation. PMA/Ionomycin is not
recommended as high control, as the usually
resulting high frequencies will not allow
conclusions on the performance of the assay
in low frequency settings.
B cells and dead cells are
gated out in FL-2 versus FL-3
according to PerCP staining and
PI staining (B). Finally, the IFN-γ
secreting T cells are shown in the
FL-1 versus FL-2 plot (C).
A detailed example for the IFN-γ
analysis is also included in the
datasheets of the Cytokine
Secretion Assays.

Vol. 4 No. 2 2000 15

 MACS NEWS

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GERMANY/AUSTRIA Miltenyi Biotec GmbH CHINA / HONG KONG Miltenyi Biotec GmbH MALAYSIA Phone: 06 33 3068
899 Dong Fang Road, Shanghai Office Biomarketing Services FAX: 06 32 0093
Friedrich-Ebert-Straße 68 Phone: +49 2204 8306-0 Holiday Inn, Room 706, Phone: 021-58305365 MEXICO Phone: 05 281 4718
51429 Bergisch Gladbach FAX: +49 2204 85197 Pudong, Shanghai 200122, FAX: 021-58305243 Uniparts S.A. FAX: 05 281 4722
e-mail: macs@miltenyibiotec.de NETHERLANDS Phone: 020 512 3202
USA/CANADA Miltenyi Biotec Inc. CLB FAX: 020 512 3570
12740 Earhart Avenue Phone: (800) FOR MACS AUSTRALIA Phone: 02 8875 7000 NORTHERN IRELAND Phone: 028 90 44 88 00
Auburn, CA 95602 Phone: (530) 888 8871 Becton Dickinson Pty. Ltd. FAX: 02 8875 7200 Ulster Anaesthetics Ltd. FAX: 028 90 44 94 00
e-mail: macs@miltenyibiotec.com FAX: (530) 888 8925 BELGIUM Phone: 03 4540066 NORWAY Phone: 02 323 3260
UNITED KINGDOM Miltenyi Biotec Ltd. SanverTECH nv FAX: 03 4541888 AH Diagnostics FAX: 02 323 3270
Almac House, Church Lane, Bisley Phone: 01483 799 800 CZECH REPUBLIC Phone: 603417315 POLAND Phone: 042 630 0736
Surrey GU24 9DR FAX: 01483 799 811 LV-Biomed FAX: 311 635157 Cytogen-Polska Sp.z.o.o FAX: 042 630 0736
e-mail: macs@miltenyibiotec.co.uk DENMARK Phone: 70 27 99 20 PORTUGAL Phone: 21 862 0550
FRANCE Miltenyi Biotec France Biotech Line AS FAX: 70 27 99 29 Citomed LDA. FAX: 21 868 4897
18 Avenue Parmentier Phone: 01 56 98 16 16 FINLAND Phone: 0892 7940200 SINGAPORE Phone: 298 4347
75011 Paris FAX: 01 56 98 16 17 Nuppulinnan Lab. FAX: 0892 81723 Biomed Diagnostics Pte. Ltd. FAX:298 4723
e-mail: macs@miltenyibiotec.fr GREECE Phone: 01 6996191 SOUTH KOREA Phone: 02 744 7859
ITALY Miltenyi Biotec S.r.l. Epsilon & Epsilon S.A FAX: 01 6925903 Fine Life Science Co. Ltd. FAX: 02 744 5281
Via Turrini, 12 Phone: 051 64 60 411 INDIA Phone: 044 220 0066 SWEDEN Phone: 031 680 490
40012 Calderara di Reno (BO) FAX: 051 64 60 499 Labmate Asia PvT Ltd. FAX: 044 220 0056 GTF FAX: 031 680 717
e-mail: macs@miltenyibiotec.it ISRAEL Phone: 03 96730 94 TAIWAN Phone: 02 273 671 00
SPAIN Miltenyi Biotec S.L. Almog Diagnostic FAX: 03 96730 91 Interlab Co. Ltd. FAX: 02 273 598 07
C/ Luis Buñuel 2 Phone: 91 512 12 90 JAPAN Phone: 03 582 094 08 TURKEY Phone: 0212 2272259
130-090-665.02

Ciudad de la Imagen FAX: 91 512 12 91 Daiichi Pure Chemicals FAX: 03 582 094 09 Laboratuar Malzemeleri FAX: 0212 2598469
28223 Pozuelo de Alarcón (M)

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