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ORIGINAL ARTICLE
In vitro-induced response patterns of antileukemic T cells:characterization by spectratyping and immunophenotyping
Susanne Reuther • Helga Schmetzer • Friedhelm R. Schuster •
Pina Krell • Christine Grabrucker • Anja Liepert • Tanja Kroell •
Hans-Jochem Kolb • Arndt Borkhardt • Raymund Buhmann
Received: 19 August 2011 / Accepted: 28 February 2012
� Springer-Verlag 2012
Abstract Myeloid leukemic cells can be induced to dif-
ferentiate into leukemia-derived dendritic cells (DCleu)
regaining the stimulatory capacity of professional DCs
while presenting the leukemic antigen repertoire. But so far,
the induced antileukemic T-cell responses are variable both
in specificity and in efficacy. In an attempt to elucidate the
underlying causes of different T-cell response patterns,
T-cell receptor (TR) Vb chain rearrangements were corre-
lated with the T cells corresponding immunophenotypic
profile, as well as their proliferative response and cytolytic
capacities. In three different settings, donor T cells, either
human leukocyte antigen matched or mismatched (haplo-
identical), or autologous T cells were repeatedly stimulated
with myeloid blasts or leukemia-derived DC/DCleus from
the corresponding patients diseased from acute myeloid
leukemia (AML). Although no significant differences in
T-cell proliferation were observed, the T-cell-mediated
cytolytic response pattern varied considerably and even
caused blast proliferation in two cases. Spectratyping
revealed a remarkable restriction ([75 % of normal level)
of the CD4? or CD8?-TR repertoire of blast- or DC/DCleu-
stimulated T cells. Although in absolute terms, DC/DCleu
stimulation induced the highest grade of restriction in the
CD8? T-cell subset, the CD4? T-cell compartment seemed
to be relatively more affected. But most importantly, in
vitro stimulation with DC/DCleu resulted into an identical
TR restriction pattern (b chain) that could be identified in
vivo in a patient sample 3 months after allo-SCT. Thus, in
vitro tests combining functional flow cytometry with
spectratyping might provide predictive information about T
cellular response patterns in vivo.
Keywords Acute myeloid leukemia � Dendritic cells �T-cell response � Immunoscope spectratyping �Immunotherapy
Introduction
Acute myeloid leukemia (AML) and myelodysplastic
syndrome (MDS) are clonal disorders of hematopoietic
stem cells, characterized by an impaired normal cell dif-
ferentiation [1]. About 70 % of successfully chemothera-
peutically treated AML patients relapse soon [2], indicating
the need for additional therapeutic strategies in order to
maintain stable remissions. So far, allogeneic stem cell
transplantation (SCT) is the only curative treatment option
[3] with donor T cells mediating the antileukemic reaction
[4, 5]. But although transfusion of donor T cells (DLT) can
re-induce complete remissions in relapsed patients after
Susanne Reuther, Helga Schmetzer, Friedhelm R. Schuster
contributed equally.
S. Reuther � F. R. Schuster � P. Krell � A. Borkhardt
Faculty of Medical, Department of Paediatric Oncology,
Haematology and Immunology, University Dusseldorf,
40225 Dusseldorf, Germany
H. Schmetzer � C. Grabrucker � A. Liepert � T. Kroell �H.-J. Kolb � R. Buhmann
Department of Medicine III, University of Munich-Grosshadern,
Munich, Germany
P. Krell
Faculty of Technology and Institute for Bioinformatics, Center
for Biotechnology, Bielefeld University, Bielefeld, Germany
H.-J. Kolb � R. Buhmann (&)
Helmholtz Center Munich, German Research Center for
Environmental Health, CCG-HCT, Marchioninistr. 25,
81377 Munich, Germany
e-mail: [email protected]
123
Clin Exp Med
DOI 10.1007/s10238-012-0180-y
SCT, there are still numerous patients who do not respond.
Moreover, appearance of graft-versus-host (GvH) reactions
can impair the efficacy of SCT or relapse therapy [3, 6].
The reasons for these varying T-cell effects have to be
elucidated. An insufficient expression of costimulatory
antigens, major histocompatibility complex (MHC) mole-
cules and tumor-associated antigens (TAA) on the surface
of cancer cells as well as disturbed mechanisms of apop-
tosis might be the main reasons for an ineffective immune
response in malignant diseases [7]. As dendritic cells (DCs)
are known to stimulate T-effector cells, especially tumor
cytotoxic T cells [8, 9], they are of potential interest for
antitumor or antileukemic vaccination strategies [10, 11].
In contrast to solid tumor cells, myeloid leukemic cells can
be converted in leukemia-derived DCs (DCleu) expressing
DC-typical antigens (e.g., CD40, CD86, CD80, CD1a and
CD83), thereby regaining the stimulatory capacity of
mature professional dendritic cells [12–14]. Thus, the
cumbersome identification and subsequent pulsing with
tumor-specific antigens probably can be overcome because
the leukemia-derived DCs might still present the complete
leukemic antigen repertoire [15–21]. In most cases, stim-
ulation with DCleu containing DC fractions (DC/DCleu)
resulted in a very effective cytotoxic T-cell response. But
there are a few cases, where opposite T-cell response
patterns have been observed, in terms of T cells mediating
anergy or even supporting blast proliferation [20–24]. So
far, these different T-cell response patterns are not pre-
dictable, but it might be expected that different ‘qualities’
of stimulators may result into different clonal compositions
and functional diversities of T-cell subsets.
Thus, in this study, we sought to determine the impact of
blast- versus DC/DCleu-mediated stimulation on T-cell
differentiation and correlated the induced T-cell receptor
(TR) b chain rearrangements with the T cells correspond-
ing immunophenotypic profile, as well as their proliferative
response and cytolytic capacities. Thereby, T-cell receptor
rearrangement analysis and functional T-cell assays might
contribute not only to the understanding of antileukemia
directed immunoreactions in myeloid leukemia, but could
also help to identify the DNA regions of T-cell receptor
loci involved in these reactions, to further isolate leukemia-
specific T cells for adoptive immunotherapy.
Materials and methods
Patient characteristics, sample collection and diagnosis
After informed consent, peripheral blood samples were
obtained from three patients with AML at the time of
diagnosis or after treatment. Diagnosis was based on the
revised World Health Organization (WHO) classification
of myeloid neoplasms and acute leukemia [25]. Cytoge-
netic analysis was performed according to standard proto-
cols and criteria defined by the International System for
Human Cytogenetic Nomenclature [26]. Patient #502 had
presented with persisting AML-M4eo after chemotherapy,
with 95 % CD34?, CD117?, CD65? and CD15? myeloid
blasts and a complex aberrant karyotype. Patient #546 had
presented with an AML-M2 at first diagnosis and 50 %
CD34?, CD117? and CD65? myeloid blasts. Patient #538
had presented with relapsed AML-M0 after SCT, normal
karyotype and 93 % CD34? and CD65? myeloid blasts
(Table 1).
DC generation
Mononuclear cells (MNC) from peripheral blood (PB) (PB-
MNC) samples were isolated by centrifugation on a Ficoll-
Hypaque (Biochrom, Berlin, Germany) density gradient,
washed and suspended in PBS without Ca2? and Mg2?
(Biochrom, Berlin, Germany). Subsequently, DCs were
generated using 3 different DC-differentiating methods
(MCM-Mimic, Picibanil and Ca-Ionophore) in parallel as
described previously [15–18, 21, 22]. The method resulting
in the best DC-counts was chosen for the quantitative
generation of DC. In brief, MNCs obtained from AML
Table 1 Sample characteristics
a Proportions of positive cells
in the mononuclear cell fraction
(MNC)
Sample no. #502 #546 #538
FAB type AML M4eo AML M2 AML MO
Karyotype 46,XY,t(2;20)qq, t(5;5)qp,
del(5p),r7, t(12;16)pq
45,X,-Y,t(8;21)qq Normal
Stage of disease Persistance First diagnosis Relapse after SCT
Leukemic blasts (%)a 95 50 93
Blast phenotype (CD) CD34, 117, 13, 33, 65, 15 CD34, 117, 33, 65, 13 CD34, 65, 33
Monocytes (%)a 2 4 2
B cells (%)a n.a. 17 3
T cells (%)a 3 8 4
NK cells (%)a 2 9 4
Clin Exp Med
123
patients in an active, blast-rich phase of the disease were
incubated in 12-well multiwell tissue culture plates in
X-Vivo 15 (BioWhittaker, Verviers, Belgium) serum-free
medium. (1) DCs were generated from 2.5 9 106 MNC/mL
in ‘MCM-Mimic’ medium containing 800 U/mL granulo-
cyte macrophage colony-stimulating factor (GM-CSF), 500
U/mL interleukin (IL)-4 and 40 ng/mL FMS-related tyro-
sine kinase 3 ligand (FLT-3). After 5 days, the same
cytokines were added to the culture again. Half medium
exchange was performed on day 8. Again GM-CSF, IL-4
and FLT-3 were added together with 150 ng/mL IL-6,
5 ng/mL IL-1b, 1 lg/mL prostaglandin PGE2 and 5 ng/mL
tumor necrosis factor TNFa. After 12 days in culture, the
DCs were harvested for subsequent experiments. (2) DCs
were generated from 7 9 105 MNC/mL in ‘Ca-Ionophore’
and were cultured in the presence of 375 ng/mL A23187
and 250 U/mL IL-4 and harvested after 3 days. (3) DCs
were generated with ‘Picibanil,’ a lysis product of Strep-
tococcus pyogenes, which has nonspecific immunmodula-
tory effects from 1 to 1.25 9 106 MNC/mL in the presence
of 500 U/mL GM-CSF and 250 U/mL IL-4. After 7 days in
culture, 5 lL/mL OK-432 (Picibanil) and 1 lg/mL PGE2
were added. The cells were harvested after 10 days in
culture. All of the substances used for DC generation are
approved for human treatment.
Flow cytometry
Flow cytometric analysis with a panel of mouse mono-
clonal antibodies (moAbs) directly conjugated with fluo-
rescein isothiocyanate (FITC), phycoerythrin (PE), tandem
Cy7-PE-conjugation (PC7) or allophycocyanine (APC)
was performed to evaluate and quantify the percentage and
phenotypes of the leukemic cells, B, T and NK cells and
DC in the PB/BM (bone marrow) samples analyzed. The
antibodies were purchased from Becton–Dickinsona (Hei-
delberg, Germany), Immunotech/Beckmann Coulterb
(Krefeld, Germany) and Caltagc (Hamburg, Germany).
CD1b(a), CD3(b), CD4(a), CD28(b), CD33(b), CD45RO(b),
CD65(b), CD83(b) and CD86(c) were used as FITC-con-
jugated moAbs. CD1a(b), CD8(a), CD56(b), CD80(b),
CD154(b) and CD206 (b) were used as PE-conjugated
moAbs. CD3(b), CD14(a), CD19(b), CD25(a), CD34(b) and
CD117(b) were chosen as the PC7-conjugated moAbs.
CD3(b), CD4(b), CD13(b), CD14(b), CD33(b), CD34(b),
CD15(a), CD45RA(a) and CD71(a) were used as APC-
conjugated moAbs. The MNC or cultured cells were sus-
pended in PBS with 20 % FCS (Biochrom, Berlin, Ger-
many) and incubated with moAbs according to the
manufacturer’s instructions. Appropriate isotype controls
were used. At least 5,000 events were evaluated on a BD
FACSCalibur flow cytometer (Becton–Dickinson, Heidel-
berg, Germany) using CellQuest data acquisition and
analysis software (Becton–Dickinson, Heidelberg, Ger-
many). For analysis and quantification of the lymphocytes,
monocytes and leukemic cells before culture the total MNC
fractions were gated. An AML sample was considered as
‘positive’ for a surface marker, if the percentage of positive
events in a gate surrounding the blasts, lymphocytes and
monocytes was more than 20 %, as described [21–23].
Proportions of positive events in defined gates compared
with the isotype controls were calculated using CellQuest
software.
Quantification and characterization of DC
For analysis and quantification of DCs and leukemia-derived
DCs, a refined gating strategy was applied and DCs were
quantified by flow cytometry as already described [20–22,
27]. In brief, for quantification, DC markers were selected,
which were not expressed on naı̈ve blasts. A refined gating
strategy was applied for an exact quantification of DC sub-
sets. A ‘blast gate’ was set surrounding the blast population
and residual lymphocytes or monocytes. The remaining cells
were gated in a ‘DC gate.’ Cells displaying the typical scatter
of dendritic cells and expressing typical DC antigens (e.g.,
CD86, CD80, CD40, CD1a and CD83) could be counted.
Moreover, ‘blasts’ could be quantified using blast-specific
markers. Blasts converted to leukemia-derived DC (DCleu)
could be quantified estimating those blast cells that had
gained a DC antigen. In addition, also blasts could be
quantified with this gating strategy that had not been con-
verted to DCleu or DC of non-leukemic origin. We also
quantified DCopt, the DC marker with highest expression
rates on DC gained after culture being not expressed on naı̈ve
blasts before. DC co-expressing CD83 was defined as
‘mature DC.’ In addition, viable DC (7AAD-/DC?) and
CCR7? DC were quantified. In AML cases with an immu-
nophenotypically detectable blast population in MNC frac-
tions, amounts of blasts being converted to DC co-expressing
specific blast antigens (e.g., CD56 and CD117) were quan-
tified. Microscopical controls were regularly performed and
revealed the DC-typical morphology of large cells with
irregular shapes and cytoplasmic projections.
Mixed lymphocyte cultures (MLC): generation
of leukemia-cytotoxic T cells
Positively selected CD3 T cells (1 9 106 cells/well) (Mil-
teney Biotech, Bergisch-Gladbach, Germany) from the
healthy stem cell donor of patient #502 and patient #538 or
autologous T cells prepared from patient #546 were cocul-
tured with irradiated (20 Gy) cell suspensions containing
25,000 DCs generated from the patient’s MNC or the same
amount of irradiated MNC as a control in 1 mL RPMI 1640
medium (Biochrom, Berlin, Germany) containing 15 %
Clin Exp Med
123
human serum (PAA Laboratories, Pasching, Austria) and 50
U/mL IL-2 (Proleukin R5, Chiron, Munich, Germany) as
described. The cells were harvested after 10 days of cocul-
ture and twofold restimulation with 5 9 104 irradiated DC/
DCleu (T*DC/DCleu) or 5x104 irradiated blasts (T*blasts) and
supplementation of IL-2, as described previously [20]. Half
medium exchange was carried out every 3–4 days. Six days
after the last restimulation, the cells were harvested and the
cytotoxicity assay was carried out.
The antigen expressions on the transferred CD3? T cells
were evaluated by FACS analyses comparing their co-
expression of CD4, CD8, CD45RA, CD45RO, CCR7 and
CD71 before and after blast and DC/DCleu stimulation.
This contributed to evaluate proportions of proliferating
(CD71?), naı̈ve, non-naive, central memory, CD4?, CD8?
or CCR7? T cells before or after blast or DC/DCleu
coculture [19, 28, 29].
Cytotoxicity assay (Fluorolysis)
The lytic activity of effector T cells was measured by a
fluorolysis assay counting viable target cells, labeled with
specific fluorochrome-labeled antibodies, before and after
effector cell (E) contact. In brief, DC/DCleu- or blast-stim-
ulated and unstimulated donor or patient-derived T cells
were cocultured in 1.5-mL Eppendorf tubes with thawed
blasts as target cells. The E:T ratio was adjusted to 1:1, and
cells were incubated for 3 h and overnight at 37 �C and 5 %
CO2. Before culture, target cells were stained for 15 min
with two FITC- and/or PE-conjugated ‘blast-’ or ‘DC-’
specific antibodies. As a control, target and effector cells
were cultured separately and mingled shortly before FACS
analysis. To evaluate amounts of viable (7AAD-) target T
cells and to quantify the cell loss after the corresponding
incubation time, cells were harvested, washed in PBS and
resuspended in a FACS flow solution containing 7AAD
(BD, Biosciences Pharmingen, Heidelberg, Germany) and a
defined number of fluorosphere beads (Becton–Dickinson,
Heidelberg, Germany). Viable, 7AAD negative cells co-
expressing specific blast marker (combinations) were
quantified taking into account defined counts of calibration
beads as described [29–31]. Cells were analyzed in a BD
FACSCalibur flow cytometer using CellQuest software
(Becton Dickinson, Heidelberg, Germany). The percentage
of lysis was the difference between proportions of viable
blasts before and after effector cell contact.
CDR3 immunoscope spectratyping of TR-Vbtranscripts
After CD4- and CD8-positive T-cell selection of the
unstimulated, or blast- or DC/DCleu-stimulated T-cell sus-
pensions on day 10 (Dynabeads; Dynal Biotech ASA, Oslo,
Norway), the total RNA was prepared from 1 9 105 cells
by using the TRIzol reagent (Invitrogen, Carlsbad, CA,
USA). Complementary DNA (cDNA) was synthesized by
random priming, using the cDNA synthesis kit (Quanti-
Tect; Qiagen, Hilden, Germany) and subjected to TR-Vbgene family-specific polymerase chain reaction (PCR) in
26 separate reactions, each containing one of the 26 Vbfamily primers in combination with a universal Cb-specific
primer [32]. In a second step, run-off products were gen-
erated from every Vb-specific PCR product by each of the
13 fluorescence-labeled, Jb-specific oligonucleotides [33].
The fragments were separated on an automated 48-capil-
lary DNA sequencer (Applied Biosystems 3730 Genetic
Analyzer; ABI, Foster City, CA, USA). The length and
fluorescence intensities of the complementary determining
region 3 (CDR3) of the different Vb–Jb combinations were
determined using Genescan 500LIZ (Applied Biosystems,
Warrington, UK) as standard and analyzed by using
GeneMapper software (Applied Biosystems, Warrington,
UK).
Cloning and sequencing of CD8-Vb13.1-Jb2.7 T-cell
receptor b chains
To sequence the CDR3 region, the cDNA derived from
CD4? and CD8? T cells was amplified by PCR in 50-lL
reaction volumes containing 19 Phusion Master Mix
(Finnzymes, Oslo, Norway) and 0.2 pmol of Vb13.1 and
Jb2.7 primer [32]. The PCR procedure included 35 cycles
of annealing at 60 �C. PCR products were purified with the
QIAquick PCR Purification Kit (Qiagen, Hilden, Ger-
many). For subsequent cloning into the TOPO TA vector
system (Invitrogen, Carlsbad, CA, USA), a 30 A-overhang
to PCR products was added to create sticky ends using
0.1–1.5 pmol of purified of PCR product, 0.2 mM dATP
(Invitrogen, Carlsbad, CA, USA) and 1 unit of Taq poly-
merase in a 50-lL reaction volume. The reaction was
incubated for 10 min at 72 �C, then placed on ice. The
TOPO TA cloning reaction and the transformation in
TOP10 chemically competent E. coli cells were performed
according to the manufacturer0s instructions. Briefly,
0.5–4 lL of the cDNA PCR product was mixed with 1 lL
of TOPO TA vector, 1 lL of salt solution (Invitrogen,
Carlsbad, CA, USA) in a 5-lL reaction volume and incu-
bated for 5 min at room temperature and then placed on ice
as preparing for the transformation in competent TOP10 E.
coli cells. 2 lL of each TOPO cloning reaction was added
to a vial of chemically competent E. coli TOP10 cells and
incubated for 10 min on ice. The cells were heat-shocked
for 30 s at 42 �C and placed on ice for 2 min. 250 lL of
S.O.C. medium was added to each vial of cells and was
shaken (200 rpm) at 37 �C for 1 h. 50 lL of each trans-
formation vial was spread out on pre-warmed culture
Clin Exp Med
123
dishes containing LB agar (Sigma-Aldrich, St. Louis, MO,
USA), 50 lg/mL Ampicillin (Sigma-Aldrich, St. Louis,
MO, USA) and 50 mg/mL 5-bromo-4-chloro-3-indolyl-b-
D-galactopyranoside (X-gal) (Promega, Madison, WI,
USA). Plates were incubated overnight at 37 �C. Trans-
formants were selected by X-gal-blue-white-screening.
Colonies containing transformants are white or light blue
colored. Colonies containing transformants were picked and
cultivated each in 3 mL LB medium (Sigma-Aldrich, St.
Louis, MO) and 3 lL Ampicillin (50 lg/mL) (Sigma-
Aldrich, St. Louis, MO, USA) overnight at 37 �C. The
Plasmid-Mini-Preparation was carried out with the Fast-
Plasmid Mini Kit (Eppendorf, Hamburg, Germany)
according to the manufacturer 0s instructions. To control the
TOPO TA cloning result, a restriction digest with EcoRI
(FastDigest, Fermentas, Leon-Rot, Germany) was per-
formed, which was analyzed by agarose gel electrophoresis.
Plasmids including the cDNA PCR product were purified
with the QIAquick PCR Purification Kit (Qiagen, Hilden,
Germany) according to the manufacturer 0s instructions.
Sequencing
The cDNA PCR products were sequenced by Sanger
reaction containing 400 ng of cDNA, 10 pmol M13 For-
ward Primer (Invitrogen, Carlsbad, CA, USA) and 4 lL of
Big Dye Sequencing RR-100 (Applied Biosytems, Foster
City, CA, USA) in a total reaction volume of 20 lL. The
PCR conditions included a first step at 96 �C (30 s), 26
cycles at 55 �C (30 s) and 60 �C (4 min). Each reaction
was purified with the DyeEx 2.0 Spin Kit (Qiagen, Hilden,
Germany) according to the manufacturer 0s instructions and
was sequenced on an Applied Biosystems 3130 genetic
analyzer (Applied Biosystems, Foster City, CA, USA).
Sequences were aligned using the VectorNTI software
(Invitrogen, Carlsbad, CA, USA).
Quantitative analysis
Spectratyping was analyzed in two ways. First, the grade of
restriction (normal and restricted) and the size of restricted
peaks were described by visual evaluation. The complexity
of restriction within the Vb families was determined by
counting the number of discrete peaks [34]. In a normal
population of T cells, CDR3 length analysis produces
approximately 5–10 identifiable or discrete peaks spaced by
3 nucleotides, with fluorescence intensity following a
Gaussian-like distribution. If discrete peaks were given but
not shown as Gaussian-like distribution, the spectratyping
profiles were determined as skewed. In cases with no discrete
peaks presented, they were scored as absent. For better
comparability, results were given also in percentages.
Taken into account that visual determination of spectra-
typing profiles suffice merely for semi-quantitative estima-
tions of clonal DNA in distinct peak positions, we performed
a second, quantitative analysis of the spectratyping data, in
order to obtain an objective and sensitive determination of
the magnitude of restriction or skewing of two given Vbfamily CDR3 profiles from a Gaussian-like distribution, the
control sample and the T-cell sample stimulated with blasts
or DC/DCleu. Therefore, the Vb-CDR3 profiles data were
analyzed using the generalized Hamming distance (HD)
analysis as introduced by Currier et al. [35, 36]. Employing a
so-called HD score, the degree of skewing of given Vb-
CDR3 Gaussian-like distributions can be analyzed, and
different distributions can be compared to one another. To
obtain the HD score for a Vb distribution with respect to a
control distribution, for each peak i (of the corresponding
Vb-CDR3 length Si), its area under the peak (Ai) relative to the
area under the whole Vb distribution (Atotal) was compared to
the corresponding value of the control, giving the degree
of skewing at each Vb-CDR3 length, Di = (Ai/Atotal) -
(Acontroli =Acontrol
total ). Summing up all absolute differences of
the frequencies of all CDR3 peaks and dividing the result
by two gives the HD score for the Vb distribution:
HD = � Ri |Di|. A Vb-CDR3 profile identical to the
control has an HD score of 0 %, while a discordant profile
obtains an HD score of 100 %. Two randomly drawn
profiles will have an HD score of 50 %.
Results
Repeated stimulation of allogeneic or autologous
T cells with myeloid blasts or blast-derived DC/DCleu
cause different T-cell response patterns
In most cases, repeated stimulation of T cells with leuke-
mic blasts or blast-derived dendritic cells result in effective
tumor cell lysis. But in some cases, T cells are generated
mediating anergy or even tumor cell growth [20, 31, 37].
To further elucidate the underlying cause for this opposite
T-cell response pattern, we studied the impact of blast- or
DC/DCleu-mediated stimulation on T-cell differentiation in
context of an autologous (#546), human leukocyte antigen
(HLA)-identical (#502) and haplo-identical (#538) experi-
mental setting. Leukemia-derived DC/DCleu from three
different patient samples (#502, #546 and #538; Table 1)
were generated as described. The results are summarized in
Table 2. In all samples tested, CD34 was identified to be
the selected marker with highest expression on blast cells
and CD206 identified to be the selected marker for the
quantification of DC since it was not expressed on naı̈ve
blasts and highest expressed on DC after culture. In patient
Clin Exp Med
123
sample #502, the Picibanil method turned out to provide
the best results: quantitative DC preparation resulted in
20 % DC, displaying in 70 % a DCleu, in 71 % a mature
and in 48 % a CCR7? DC phenotype. In patient sample
#546, we could generate 21 % DC with Picibanil display-
ing in 45 % DCleu, in 45 % a mature and in 2 % a CCR7?
DC phenotype. In patient sample #538, we succeeded to
generate 15 % DC with MCM-Mimic being the best
method, displaying in 62 % a DCleu, in 48 % a mature and
in 61 % a CCR7? DC phenotype.
To determine the impact of these different stimulator
cells (blasts or DC/DCleu) on the T-cell response pattern,
MLC were set up with the corresponding donor T cells
(#502, HLA-identical (sister); #538, haplo-identical
(mother); #546, (autologous). After a 10-day stimulation
period with blasts or DC/DCleu, respectively, T cells were
harvested and phenotypically characterized as shown in
Fig. 1. In all three cases, more than 95 % viable, 7AAD-
negative T cells were found after stimulation. T-cell pro-
liferation, as to be measured by co-expression of CD71,
increased in all cases during the blast and DC/DCleu
stimulation period. With the exception of patient #546,
where CD45RO? T cells declined to 15 % after blast
stimulation, CD45RO? non-naive T cells increased after
blast and even to a higher extent after DC/DCleu stimula-
tion. Moreover, in the allogeneic setting (patient #502,
patient #538), stimulation with myeloid blasts resulted in
increased proportions of CD8? T cells, whereas stimulation
with DC/DCleu seemed to favor the increase in CD4? T
cells. Although the percentage of T cells expressing che-
mokine receptors varied in the settings compared, stimu-
lation with DC/DCleu caused increased CCR4 expression
levels (Fig. 1). In an attempt to compare the induced T-cell
activities, cytotoxicity assays were performed. In patient
#502, we observed that both, blast- and DC/DCleu-stimu-
lated T cells mediated blast lysis. But interestingly, stim-
ulation with DC/DCleu resulted not only in a more
effective, but also in a more prolonged lytic activity, as
measured after a period of 3 and 24 h of incubation with
target cells (Fig. 2). However, in marked contrast, in
patient #546 and #538, we observed that both, blast- and
DC/DCleu-stimulated T cells resulted in blast proliferation
(Fig. 2). In previous experiments, we found that a
CD4:CD8 ratio of [1 after DC/DCleu stimulation corre-
lated with a cytotoxic activity of the effector cell popula-
tion [24]. In case #502, the calculated CD4:CD8 ratio was
3.1 after DC/DCleu stimulation, and in cases #546 and
#538, the calculated ratio was 1.1 and 0.9, further sup-
porting this previous observation. No correlations could be
established for unstimulated or blast-stimulated T cells.
Blast or DC/DCleu stimulation results into distinctive
TR-Vb profiles
To further dissect the stimulatory impact on the effector
cells as well as to evaluate changes in the amounts of gene
products in Vb subunits in different settings, TR-Vb gene
profiles were analyzed via immunoscope spectratyping.
Results obtained in cases #502, #546 and #538 are sum-
marized in Table 3 and Fig. 3. The restricted repertoires
are highlighted with red circles (Fig. 3). In all three cases,
CD8? T cells showed a more restricted TR repertoire than
CD4? T cells after stimulation, independently of blast or
DC/DCleu used as stimulators. Moreover, with exception of
Table 2 Generation of
leukemia-derived dendritic cells
(DCleu)
DCleu/MNC: percentage of
DCleu in the MNC fraction
DCleu/DC: percentage of DCleu
in the DC fraction
Sample no. #502 #546 #538
A) Determination of the best DCleu conversion conditions
Selected marker profile
Blast CD34 34 34 34
DC CD206 206 206 206
DC pre-culture
(% DC generated) MCM-Mimic 13 15 15
Picibanil 24 21 n.a.
Ca-Ionophore 5 19 7
B) Quantitative results of the DCleu preparation
DC/MNC (%) 20 21 15
Blasts converted/MNC (%) 25 25 27
DCleu/MNC (%) 10 5 5
DCleu/DC (%) 70 45 62
Viable DC/DC (%) 88 75 75
Mature DC/DC (%) 71 45 48
Migratory DC/DC (%) 48 2 61
Clin Exp Med
123
DC/DCleu-stimulated CD4? T cells from patient #538,
stimulation with DC/DCleu resulted in a higher grade of
restriction than stimulation with blasts (Table 3).
In patient #502, the unstimulated CD4? and CD8? T
lymphocytes derived from the corresponding HLA-matched
donor displayed a normal, non-restricted Gaussian-like
Stimulation with myeloid blasts Stimulation with DC/DCleu
patient #502
patient #546
patient #538
Fig. 1 Positively selected CD3 T cells from the healthy stem cell
donors of patient #502 and patient #538 or autologous T cells
prepared from patient #546 were cocultured for 10 days with
irradiated blasts or DC/DCleu. Subsequently, the antigen expressions
of the CD3 T cells were evaluated by flow cytometry and compared
for their co-expression of CD4, CD8, CD45RA, CD45RO, CCR7 and
CD71 before and after blast and DC/DCleu stimulation. This
contributed to evaluate distinct proportions of proliferating
(CD71?), naı̈ve, non-naı̈ve and central memory T cells in context
of the respective stimulation
Clin Exp Med
123
distribution of the CDR3 region of all Vb–Jb gene com-
binations of the TR, indicating a normal TR repertoire
(Fig. 3a). After blast or DC/DCleu stimulation, both the
CD4? and the CD8? T-cell subset displayed a significant
TR restriction, but more pronounced for the CD8? T-cell
subset (56/30 peaks in the CD8*blast/CD8*DC/DCleu fraction
vs. 88/36 peaks in the CD4*blast/CD4*DC/DCleu fraction).
Interestingly, the DC/DCleu stimulation induced an even
more skewed TR pattern, affecting both the CD4? and
CD8? T-cell subsets (88 peaks in the CD4*blast fraction vs.
36 peaks in the CD4*DC/DCleu fraction, and 56 peaks in the
CD8*blast fraction versus 30 peaks in the CD8*DC/DCleu
fraction). Moreover, the percental distribution of clonal
DNA showed highest amounts of Vb5.2 DNA (13.9 %)
and 8.3 % of Vb2, Vb5.1, Vb8 and Vb18 DNA in the
CD4*DC/DCleu fractions, whereas the percentages of clonal
DNA stayed B6.8 % in the CD4*blast-stimulated T-cell
subsets (Table 3). Concerning the percental distribution of
clonal DNA in the CD8*DC/DCleu fractions, highest amounts
were found in the Vb18 DNA (13.3 %) and each 10 % in
the Vb5.2, Vb10 and Vb23 DNA, whereas the values
stayed B8.9 % in the CD8*blast fraction (Table 3).
Cytotoxicity after 3 hours Cytotoxicity after 24 hours
patient #502
patient #546
patient #538
Fig. 2 Cytolytic activity of unstimulated versus blast- or DC/DCleu-
stimulated allogeneic (#502; #538) or autologous (#546) T cells.
Antileukemic activity was evaluated via a non-radioactive fluorolysis
assay after 3-and 24-h periods of co-incubation of the corresponding
effector cells and blasts. Positive bars indicate blast lysis, and
negative values display blast growth
Clin Exp Med
123
Ta
ble
3V
bp
eak
sas
det
ecte
db
yim
mu
no
sco
pe
spec
trat
yp
ing
wit
hin
the
TR
gen
elo
cus
bef
ore
and
afte
rst
imu
lati
on
of
Tce
lls
wit
hm
yel
oid
bla
sts
or
DC
/DC
leu
Vb
1V
b2
Vb
3V
b4
Vb5
.1V
b5
.2V
b6
Vb
7V
b8
Vb
9V
b1
0V
b1
1V
b1
2V
b13
.1
Pat
ien
t#
50
2
CD
4d
ay0
3(2
.1)
7(5
.0)
5(3
.6)
5(3
.6)
4(2
.9)
7(5
.0)
6(4
.3)
6(4
.3)
6(4
.3)
7(5
.0)
3(2
.1)
7(5
.0)
4(2
.9)
7(5
.0)
CD
4b
last
day
10
2(2
.3)
5(5
.7)
3(3
.4)
3(3
.4)
4(4
.6)
5(5
.7)
5(5
.7)
4(4
.6)
3(3
.4)
3(3
.4)
2(2
.3)
3(3
.4)
2(2
.3)
6(6
.8)
CD
4D
C/D
Cle
ud
ay1
00
3(8
.3)
2(5
.6)
2(5
.6)
3(8
.3)
5(1
3.9
)2
(5.6
)0
3(8
.3)
1(2
.8)
1(2
.8)
1(2
.8)
1(2
.8)
1(2
.8)
CD
8d
ay0
4(3
.4)
7(5
.9)
5(4
.2)
4(3
.4)
1(0
.8)
5(4
.2)
5(4
.2)
5(4
.2)
6(5
.1)
6(5
.1)
1(0
.8)
5(4
.1)
4(3
.4)
6(5
.1)
CD
8b
last
day
10
3(5
.4)
4(7
.1)
2(3
.6)
2(3
.6)
1(1
.8)
3(5
.4)
4(7
.1)
2(3
.6)
2(3
.6)
3(5
.4)
2(3
.6)
2(3
.6)
1(1
.8)
4(7
.1)
CD
8D
C/D
Cle
ud
ay1
01
(3.3
)1
(3.3
)1
(3.3
)1
(3.3
)1
(3.3
)3
(10
.0)
1(3
.3)
00
1(3
.3)
3(1
0.0
)0
01
(3.3
)
Pa
tien
t#
54
6
CD
4d
ay0
1(3
.7)
01
(3.7
)0
00
2(7
.4)
1(3
.7)
2(7
.4)
2(7
.4)
01
(3.7
)0
2(7
.4)
CD
4b
last
day
10
3(3
.1)
3(3
.1)
6(6
.1)
2(2
.0)
3(3
.1)
6(6
.1)
5(5
.1)
4(4
.1)
4(4
.1)
6(6
.1)
1(1
.0)
2(2
.0)
3(3
.1)
5(5
.1)
CD
4D
C/D
Cle
ud
ay1
01
(2.6
)2
(5.3
)1
(2.6
)1
(2.6
)3
(7.9
)1
(2.6
)2
(5.3
)1
(2.6
)3
(7.9
)1
(2.6
)3
(7.9
)0
02
(5.3
)
CD
8d
ay0
02
(5.0
)1
(2.5
)1
(2.5
)0
2(5
.0)
3(7
.5)
4(1
0.0
)5
(12
.5)
2(5
.0)
1(2
.5)
00
1(2
.5)
CD
8b
last
day
10
1(1
.9)
1(1
.9)
2(3
.9)
1(1
.9)
03
(5.8
)4
(7.7
)5
(9.6
)5
(9.6
)2
(3.9
)2
(3.9
)0
03
(5.8
)
CD
8D
C/D
Cle
ud
ay1
01
(2.9
)2
(5.9
)1
(2.9
)2
(5.9
)1
(2.9
)1
(2.9
)3
(8.8
)2
(5.9
)2
(5.9
)4
(11
.8)
00
04
(11
.8)
Pat
ien
t#
53
8
CD
4d
ay0
5(1
7.8
)1
(3.6
)2
(7.1
)2
(7.1
)0
01
(3.6
)5
(17
.8)
03
(10
.7)
3(1
0.7
)0
00
CD
4b
last
day
10
1(3
.1)
3(9
.4)
2(6
.3)
00
04
(12
.5)
4(1
2.5
)4
(12
.5)
00
00
3(9
.4)
CD
4D
C/D
Cle
ud
ay1
00
1(2
.1)
1(2
.1)
00
2(4
.3)
4(8
.5)
1(2
.1)
1(2
.1)
3(6
.4)
04
(8.5
)4
(8.5
)3
(6.4
)
CD
8d
ay0
1(8
.3)
2(1
6.6
)2
(16
.6)
00
2(1
6.6
)2
(16
.6)
01
(8.3
)0
00
00
CD
8b
last
day
10
00
2(1
2.5
)2
(12
.5)
00
01
(6.3
)0
01
(6.3
)0
05
(31
.3)
CD
8D
C/D
Cle
ud
ay1
00
02
(28
.6)
00
00
01
(14
.3)
00
00
2(2
8.6
)
Vb
13
.2V
b1
4V
b1
5V
b1
6V
b1
7V
b1
8V
b19
Vb
20
Vb
21
Vb
22
Vb
23
Vb
24
Sco
reS
core
(%)
Rat
io
Pat
ien
t#
50
2
CD
4d
ay0
6(4
.3)
6(4
.3)
5(3
.6)
6(4
.3)
6(4
.3)
5(3
.6)
1(0
.7)
4(2
.9)
6(4
.3)
7(5
.0)
5(3
.6)
6(4
.3)
14
01
00
CD
4b
last
day
10
5(5
.7)
5(5
.7)
3(3
.4)
3(3
.4)
5(5
.7)
4(4
.6)
1(1
.1)
02
(2.3
)5
(5.7
)4
(4.6
)1
(1.1
)8
86
32
.4
CD
4D
C/D
Cle
ud
ay1
00
1(2
.8)
01
(2.8
)1
(2.8
)3
(8.3
)0
01
(2.8
)2
(5.6
)1
(2.8
)1
(2.8
)3
62
6
CD
8d
ay0
5(4
.1)
7(5
.9)
3(2
.5)
6(5
.1)
6(5
.1)
3(2
.5)
5(4
.1)
1(0
.8)
3(2
.5)
6(5
.1)
5(4
.2)
5(4
.1)
11
91
00
CD
8b
last
day
10
2(3
.6)
5(8
.9)
01
(1.8
)5
(8.9
)0
00
2(3
.6)
3(5
.4)
3(5
.4)
05
64
71
.9
CD
8D
C/D
Cle
ud
ay1
01
(3.3
)0
2(6
.7)
01
(3.3
)4
(13
.3)
00
2(6
.7)
1(3
.3)
3(1
0.0
)2
(6.7
)3
02
5
Pat
ien
t#
54
6
CD
4d
ay0
04
(14
.8)
00
3(1
1.1
)1
(3.7
)2
(7.4
)1
(3.7
)0
2(7
.4)
1(3
.7)
1(3
.7)
27
10
0
CD
4b
last
day
10
)5
(5.1
)s
(5.1
)4
(4.1
)3
(3.1
)3
(3.1
)3
(3.1
)1
(1.0
)4
(4.1
)1
(1.0
)6
(6.1
)5
(5.1
)5
(5.1
)9
83
62
2.6
CD
4D
C/D
Cle
ud
ay1
00
5(1
3.2
)0
01
(2.6
)2
(5.3
)1
(2.6
)2
(5.3
)0
2(5
.3)
2(5
.3)
2(5
.3)
38
14
0
CD
8d
ay0
02
(5.0
)1
(2.5
)1
(2.5
)3
(7.5
)2
(5.0
)1
(2.5
)2
(5.0
)0
2(5
.0)
3(7
.5)
1(2
.5)
40
10
0
CD
8b
last
day
10
3(5
.8)
1(1
.9)
1(1
.9)
3(5
.8)
2(3
.9)
3(5
.8)
02
(3.9
)0
1(1
.9)
4(7
.7)
3(5
.8)
52
13
01
.5
Clin Exp Med
123
The autologous T lymphocytes of case #546 were col-
lected at first diagnosis and presented with a restricted TR-
Vb repertoire in CD4? and CD8? T-cell subset repertoire
(Fig. 3b). After stimulation an increase of the TR reper-
toire was observed. But again, stimulation with DC/DCleu
correlated with a higher grade of TR restriction than
stimulation with blasts and affected to a higher extend
the CD8? T-cell subset (98 peaks in the CD4*blast frac-
tion versus 38 peaks in the CD4*DC/DCleu fraction, and 52
peaks in the CD8*blast fraction versus 34 peaks in the
CD8*DC/DCleu fraction). The percental distribution of the
clonal DNA showed 13.2 % of Vb14 and high amounts of
Vb5.1, Vb8 and Vb10 (7.9 % each) after DC/DCleu stim-
ulation of CD4? T cells, whereas the percentages of clonal
DNA stayed B6.1 % in the blast-stimulated subset. Perc-
ental distributions of clonal DNA in the CD8? T-cell
fraction showed 9.6 % in Vb7 and Vb8 DNA after blast
stimulation. After DC/DCleu stimulation, the CD8? T-cell
fraction was found to have highest restricted DNA clones
in Vb9 and Vb13.1 (11.8 % each) and Vb6 and Vb24
(8.8 % each).
Unstimulated CD4? and CD8? T lymphocytes derived
from the HLA haplo-identical donor of patient #538
showed in both T-cell subsets a normal Gaussian-like
distribution of the Vb family TR repertoire (Fig. 3c). After
blast stimulation, the CD4? T-cell subset revealed a
slightly more restricted Vb-TR pattern as compared to the
CD4? T-cell subset stimulated with DC/DCleu (32 peaks in
the CD4*blast fraction vs. 47 peaks in the CD4*DC/DCleu
fraction). The CD8? T-cell subset, however, showed again
a more restricted Vb repertoire after DC/DCleu stimulation
(16 peaks in the CD8*blast fraction vs. 7 peaks in the
CD8*DC/DCleu fraction). The percental distribution in the
CD4 subset after blast stimulation showed highest amounts
of clonal DNA for Vb16 (15.6 %) and Vb6, Vb7 and Vb8
DNA (12.5 % each). After DC/DCleu stimulation, DNA
was distributed to Vb14 (10.6 %), and Vb6, Vb11, Vb12
and Vb24 (8.5% each). Within the CD8*blast fraction,
clonal DNA was highly distributed to Vb13.1 (31.3 %),
and Vb3, Vb4 and Vb18 (12.5 % each). After DC/DCleu
stimulation, Vb3 and Vb13.1 are presented with 28.6 %
and Vb8, Vb18 and Vb22 with 14.3 % of the clonal DNA
(Table 3). In summary, the strongest Vb-TR restriction
was found within the CD8? T-cell subset after stimulation
with DC/DCleu, independent of the functional activity of
the involved T cells in all three patients.
Hamming distance displays disparity of the Vb profile
restriction in dependence of blast or DCleu stimulation
To quantify the degree of Vb-TR restriction also in an
objective and sensitive manner, Hamming distance (HD)
scores were calculated for the determined spectratypeTa
ble
3co
nti
nu
ed
Vb
13
.2V
b1
4V
b1
5V
b1
6V
b1
7V
b1
8V
b19
Vb
20
Vb
21
Vb
22
Vb
23
Vb
24
Sco
reS
core
(%)
Rat
io
CD
8D
C/D
Cle
ud
ay1
00
1(2
.9)
02
(5.9
)0
01
(2.9
)0
02
(5.9
)2
(5.9
)3
(8.8
)3
48
5
Pat
ien
t#
53
8
CD
4d
ay0
1(3
.6)
1(3
.6)
00
01
(3.6
)0
03
(10
.7)
00
01
36
10
0
CD
4b
last
day
10
00
1(3
.1)
5(1
5.6
)0
01
(3.1
)1
(3.1
)0
02
(6.3
)1
(3.1
)3
22
40
.7
CD
4D
C/D
Cle
ud
ay1
03
(6.4
)5
(10
.6)
00
1(2
.1)
03
(6.4
)3
(6.4
)0
3(6
.4)
1(2
.1)
4(8
.5)
47
35
CD
8d
ay0
00
01
(8.3
)0
1(8
.3)
00
00
00
12
91
00
CD
8b
last
day
10
1(6
.3)
00
1(6
.3)
02
(12
.5)
00
00
1(6
.3)
01
61
22
.3
CD
8D
C/D
Cle
ud
ay1
00
00
00
1(1
4.3
)0
00
1(1
4.3
)0
07
5
Pea
kco
un
tsw
ith
inV
bfa
mil
ies
of
po
siti
vel
yse
lect
edC
D4
?an
dC
D8
?T
cell
sar
ep
rese
nte
db
efo
re(‘
day
0’)
or
afte
rst
imu
lati
on
for
10
day
s(‘
day
10
’).P
eak
so
fea
chV
bfa
mil
yar
ep
rese
nte
din
nu
mb
ers,
resp
ecti
vel
y,
by
cou
nti
ng
all
pea
ks
for
each
Vb
fam
ily
inb
rack
ets
asp
erce
nta
ge
refe
rrin
gto
the
tota
ln
um
ber
of
pea
ks.
Th
eo
ver
all
cou
nts
of
the
pea
ks
are
sum
mar
ized
asa
sco
re.
Th
e
rati
oo
fb
last
and
DC
/DC
leu
stim
ula
tio
nex
pre
sses
the
rela
tiv
ere
stri
ctio
no
fth
eT
Rre
per
toir
ew
ith
inth
eco
rres
po
nd
ing
T-c
ell
sub
set
Th
eb
old
face
dv
alu
esin
dic
ate
the
Vb
fam
ilie
sw
ith
the
hig
hes
tp
eak
cou
nts
and
corr
esp
on
din
gp
erce
nta
ge
val
ues
wit
hin
the
spec
ific
frac
tio
ns
Clin Exp Med
123
(A) patient #502
CD4+ T cell fractions day 0 (w/o stimulation) day 10 (stimulation blast) d10 (DC/DCleustimulation
base pair position →
CD8+ T cell fractions
day 0 (w/o stimulation) day 10 (stimulation blast) day 10 (DC/DCleustimulation
base pair position →
fluo
resc
ence
inte
nsit
y →
fluo
resc
ence
inte
nsit
y →
Fig. 3 T-cell receptor Vb families 3 and 13.1 with the corresponding
Jb families (1.1–2.7), as determined by immunoscope spectratyping,
are shown for the selected CD4? and CD8? T cells derived from
patients #502, #546 and #538, before and after 10 days of in vitro
stimulation with either blasts or DC/DCleu. The line on the top shows
both Vb3 (first Gaussian-like distribution) and Vb13.1 (second
Gaussian-like distribution) families. The Vb3 family peaks are
located between the 160 and 200 base pair position, whereas the
Vb13.1 family exhibits a base pair length of 220–240. The lines
below show the corresponding Jb families labeled with three different
colors: The second line presents the Jb families 1.1 (green), 1.2 (blue)
and 1.3 (black). In the third line, the Jb families 1.4 (green), 1.5 (blue)
and 1.6 (black) are presented. The forth line involves the Jb families
2.1 (green), 2.2 (blue) and 2.3 (black). In the fifth line, the Jb families
2.4 (green), 2.5 (blue) and 2.6 (black) are shown, and in the bottom
line, the Jb family 2.7 (green) is presented. The red circles indicate
the development of a highly restricted repertoire after both stimula-
tion settings, either with blasts or DCleu (color figure online)
Clin Exp Med
123
profiles after blast and DCleu stimulation, respectively, and
compared with the corresponding internal control distri-
bution (day 0 w/o stimulation) spanning the total range of
CDR3 lengths.
As exemplarily shown in Fig. 4a (patient #502), HD
scores for the CD4? TR-Vb3 and Vb13.1 family were
calculated to be 38 % and 13 % after blast stimulation, and
46 % and 47 % after DCleu stimulation, respectively,
indicating a higher degree of restriction after DCleu stim-
ulation. Within the CD8? T-cell fraction, HD scores for the
corresponding families were determined to be 51 and 36 %
after blast stimulation, and 50 and 72 % after DCleu stim-
ulation. According to the calculation formula, HD scores of
50 % indicate absence of the Vb profile as compared with
Patient #546 CD4+ T cell fractions
day 0 (w/o stimulation) day 10 (stimulation blast) day 10 (DC/DCleustimulation)
base pair position →
CD8+ T cell fractions day 10 (stimulation blast)day 0 (w/o stimulation) day 10 (DC/DCleustimulation)
base pair position →
fluo
resc
ence
inte
nsit
y →
fluo
resc
ence
inte
nsit
y →
(B)
Fig. 3 continued
Clin Exp Med
123
the internal control, whereas HD scores of 36 % indicate
intermediate, 51 % strong and 72 % very strong restriction
of the corresponding Vb profiles.
In patient #546 (Fig. 4b), the HD scores for the Vb3 and
Vb13.1 families within the CD4? fraction were calculated
to be 70 and 40 % after stimulation with blasts, and 57 and
16 % after DCleu stimulation. For the CD8? subpopulation,
HD scores for the corresponding families were 31 and
48 % after blast stimulation and 0 and 16 % after DCleu
stimulation, whereas according to calculation formula, an
HD score of 0 % is indicating an identical Vb3 profile, as
compared to the internal control (day 0 w/o stimulation).
Although in this case, the internal control displayed already
a skewed Vb repertoire, further stimulation with DCleu
Patient #538
CD4+ T cell fractions
base pair position →CD8+ T cell fractions
day 0 (w/o stimulation) day 10 (stimulation blast) leustimulation) day 10 (DC/DC
day 0 (w/o stimulation) day 10 (stimulation blast) day 10 (DC/DCleustimulation)
base pair position →
fluo
resc
ence
inte
nsit
y →
fluo
resc
ence
inte
nsit
y →
(C)
Fig. 3 continued
Clin Exp Med
123
resulted in a more restricted TR-Vb profile as compared to
stimulation with blasts.
For patient #538 (Fig. 4c), the corresponding HD scores
for the CD4? TR-Vb3 and Vb13.1 family were calculated
to be 33 % and 32 % after blast stimulation, and 64 % and
58 % after DCleu stimulation, respectively, indicating a
more skewed repertoire after DCleu stimulation. Within the
CD8? compartment, HD scores for the corresponding
Patient #502day 0 (w/o stimulation) day 10 (stimulation blast) d10 (DC/DCleustimulation)
Vβ 3 Vβ 13.1 Vβ 3 Vβ 13.1 Vβ 3 Vβ 13.1
CD4+
HD score control control 38 % 13 % 46 % 47 %
CD8+
HD score control control 51 % 36 % 50 % 72 %
Patient #546
day 0 (w/o stimulation) day 10 (stimulation blast) d10 (DC/DCleustimulation)
Vβ 3 Vβ 13.1 Vβ 3 Vβ 13.1 Vβ 3 Vβ 13.1
CD4+
HD score control control 70 % 40 % 57 % 16 %
CD8+
HD score control control 31 % 48 % 0 % 16 %
Patient #538day 0 (w/o stimulation) day 10 (stimulation blast) d10 (DC/DCleustimulation)
Vβ 3 Vβ 13.1 Vβ 3 Vβ 13.1 Vβ 3 Vβ 13.1
CD4+
HD score control control 33 % 32 % 64% 58 %
CD8+
HD score control control 88 % 68 % 32 % 38 %
(A)
(B)
(C)
Fig. 4 Hamming distance scores were calculated for the Vb3 and
Vb13.1 Gaussian-like distributions of all three patients to obtain the
extent of perturbation or skewing at each CDR3 length. The HD score
for each Vb distribution with respect to a control distribution, for each
peak i (of the corresponding Vb-CDR3 length Si), its area under the
peak (Ai) relative to the area under the whole Vb distribution (Atotal)
was compared to the corresponding value of the control, giving the
degree of skewing at each Vb-CDR3 length, Di = (Ai/Atotal) -
(Acontroli =Acontrol
total ). Summing up all absolute differences of the
frequencies of all CDR3 peaks and dividing the result by two gives
the HD score for the Vb distribution: HD = � Ri |Di|
Clin Exp Med
123
families were found to be 88 and 68 % after blast stimu-
lation, and 32 and 38 % after DCleu stimulation, indicating
a more pronounced TR restriction for the Vb3 and Vb13.1
family after blast stimulation. However, HD score analysis
of the additional Vb families of the CD8 compartment
(data not shown) again supported the observation that
DCleu stimulation resulted in a more restricted Vb profile
than blast stimulation.
Blast or DC/DCleu stimulation results into distinctive
TR-Jb profiles
Additionally, TR-Jb gene profiles were analyzed via im-
munoscope spectratyping. The Jb peak score ranges for an
unstimulated T-cell population between 1,000 and 1,400 Jbpeaks. In all three cases, a restricted Jb repertoire was
observed after blast or DC/DCleu stimulation as summa-
rized in Table 4. With exception of case #538 (337 peaks
in the CD4*blast fraction vs. 569 peaks in the CD4*DC/DCleu
fraction and 344 peaks in the CD8*blast fraction vs. 528
peaks in the CD8*DC/DCleu fraction), again the CD8? T-cell
subset displayed a higher grade of TR restriction as com-
pared to the CD4? T-cell subset. Moreover, stimulation
with DC/DCleu was superior to stimulation with blasts and
caused a more restrictive Jb repertoire in both T-cell sub-
sets. Regarding case #502, the percental distribution of
clonal Jb DNA families showed highest amounts of Jb2.3
and 2.7 in all CD4? and CD8? T-cell fractions. In case
#546, it is shown that the Jb2.3 and Jb2.7 DNA families
were often recombined within their TR b chains in all
CD4? and CD8? T-cell fractions as well. Concerning case
#538, it is shown that the Jb family 2.7 is recombined in all
CD4? and CD8? T-cell fractions for the most part.
Donor T cells stimulated in vitro with DC/DCleu give
rise to one T-cell clone that could also be found in vivo
after allogeneic transplantation
In an attempt to evaluate whether in vitro stimulation might
provide predictive information about potential TR restric-
tion patterns evolving after transplantation, T cells derived
3 months after SCT from patient #538 were analyzed and
compared with the Vb profiles of CD4? and CD8? T cells
as obtained after in vitro stimulation with DC/DCleu. As
shown and exemplified for the Vb3 and Vb13.1 families in
Fig. 5, spectratyping of T cells obtained 96 days after stem
cell transplantation displayed restricted peaks located at the
same base pair positions as found after in vitro stimulation
with DC/DCleu. To ascertain clonal identity, specific
Vb13.1/Jb2.7 PCR products were subcloned and sequence
analysis performed. Out of 220 different CD8? Vb-rear-
ranged T-cell clones generated, screening for identical
sequences revealed the same sequence once in an in vivo
and tenfold in in vitro CD8? T-cell clones after DC/DCleu
stimulation (Fig. 6). Thus, T cells stimulated in vitro with
DC/DCleu gave rise to identical, specifically TR rearranged
T-cell clones as found in vivo after allogeneic stem cell
transplantation.
Discussion
Allogeneic SCT provides a potential curative treatment
option in myeloid leukemia, taking advantage of a graft-
versus-leukemia (GvL) effect mediated by donor lympho-
cytes [3]. Even in recurrent myeloid leukemia after allo-
geneic SCT, subsequent transfusion of donor lymphocytes
can induce sustained remissions in some patients [4, 6]. But
so far, the prerequisite conditions to induce a productive,
leukemia-directed immune response in vivo are not well
understood.
Taken the central role of dendritic cells in adaptive
immunity, we already demonstrated the potential of leu-
kemia-derived dendritic cells (DCleu) to put forward an
effective antileukemic T-cell response. However, in a few
cases, T-cell clones mediating anergy or even blast pro-
liferation could be observed and it already became obvious
that both quantity and quality of the generated DCleus as
well as the induced microenvironment might be crucial for
the induction of a protective T-cell response [20, 31, 37].
In the present report, we tried to further characterize and
delineate the impact of blast- versus DCleu-mediated
stimulation on the evolving T-cell response in context of an
autologous or allogeneic setting. Myeloid blasts were
derived from three patients with active disease before or
after SCT and successfully induced to differentiate in
myeloid leukemia-derived dendritic cells as published
previously [18, 21, 37]. Quantitative conversion of blasts
resulted in 15–21 % DCs, with 45–70 % of these DCs
being characterized to be of leukemic origin (DC/DCleu).
Stimulation with DC/DCleu did not further extend the
T-cell proliferation (range 43–49 %) in the allogeneic
context; however, it was found to be strikingly superior in
the autologous system (#546) as compared to stimulation
with blasts (41 vs. 16 %; Fig. 1), indicating the gain of
immunostimulatory competence of myeloid blasts via
conversion. Thereby, stimulation with DC/DCleu favored
the expansion of CD4?, CCR4? and CD45RO? T cells. In
previous publications, it was hypothesized that a CD4:CD8
T cell ratio [1 after DC/DCleu stimulation might be pre-
dictive and permissive for the induction of an effective
tumor cell lysis [23, 24]. In line with this hypothesis, case
#502 displayed a significant tumor cell lysis. But interest-
ingly, both DC/DCleu- and blast-stimulated T cells resulted
in effective blast lysis. However, DC/DCleu-stimulated T
cells seemed to mediate not only a more efficient lysis than
Clin Exp Med
123
Ta
ble
4Jb
pea
ks
det
ecte
db
yim
mu
no
sco
pe
spec
trat
yp
ing
of
CD
4?
and
CD
8?
Tce
lls
wer
eco
un
ted
for
each
Jbfa
mil
y,
resp
ecti
vel
y,
bef
ore
(‘d
ay0
’)an
daf
ter
stim
ula
tio
n(‘
day
10
’)w
ith
my
elo
idb
last
so
rD
C/D
Cle
uto
ob
tain
the
sem
i-q
uan
tita
tiv
ean
aly
sis
asp
erfo
rmed
for
the
Vb
fam
ilie
s
Jb1.1
Jb1.2
Jb1.3
Jb1.4
Jb1.5
Jb1.6
Jb2.1
Jb2.2
Jb2.3
Jb2.4
Jb2.5
Jb2.6
Jb2.7
Sco
reS
core
(%)
Rat
io
Pat
ient
#502
CD
4day
099
(9.1
)57
(5.1
)92
(8.2
)22
(2.1
)63
(5.2
)41
(3.2
)114
(10.2
)97
(9.1
)154
(14.1
)26
(2.1
)114
(10.2
)65
(6.1
)127
(11.2
)1071
100
CD
4bla
stday
10
49
(4.2
)45
(4.1
)37
(3.2
)36
(3.1
)45
(4.1
)28
(2.2
)38
(3.2
)23
(2.1
)69
(6.1
)26
(2.1
)51
(4.2
)19
(1.2
)76
(7.1
)542
51
3
CD
4D
C/D
Cle
uday
10
22
(2.1
)6
(0.2
)21
(1.2
)7
(0.2
)3
(0.1
)9
(0.2
)18
(1.2
)0
24
(2.1
)14
(1.1
)15
(1.1
)7
(0.2
)17
(1.2
)163
15
CD
8day
081
(7.2
)57
(5.1
)88
(8.1
)45
(4.1
)57
(5.1
)43
(4.1
)119
(11.1
)50
(4.2
)132
(12.1
)40
(3.2
)26
(2.1
)15
(1.1
)74
(6.2
)827
100
CD
8bla
stday
10
18
(1.2
)21
(1.2
)19
(1.2
)11
(1.1
)9
(0.2
)13
(1.1
)47
(4.1
)16
(1.1
)49
(4.2
)10
(0.2
)17
(1.2
)9
(0.2
)25
(2.1
)264
32
1.9
CD
8D
C/D
Cle
uday
10
1(1
.1)
1(1
.1)
8(0
.2)
1(0
.1)
1(0
.1)
8(0
.2)
21
(1.2
)12
(1.1
)23
(2.1
)10
(0.2
)17
(1.2
)2
(0.1
)15
(1.1
)140
17
Pat
ient
#546
CD
4day
089
(6.2
)112
(8.2
)105
(7.2
)72
(5.1
)86
(6.2
)93
(7.1
)117
(8.2
)83
(6.1
)97
(7.1
)113
(8.2
)127
(9.2
)75
(5.2
)148
(11.1
)1317
100
CD
4bla
stday
10
91
(6.2
)71
(5.1
)88
(6.2
)55
(4.1
)56
(4.1
)59
(4.1
)78
(5.1
)46
(3.1
)96
(7.1
)35
(2.2
)102
(7.2
)54
(4.1
)21
(1.2
)852
65
4.2
CD
4D
C/D
Cle
uday
10
17
(1.1
)26
(1.2
)20
(1.2
)16
(1.1
)9
(0.2
)18
(1.1
)15
(1.1
)4
(0.1
)22
(1.2
)10
(0.2
)19
(1.1
)5
(0.1
)24
(1.2
)205
16
CD
8day
067
(5.1
)94
(7.1
)99
(7.2
)86
(6.2
)75
(5.2
)121
(9.1
)145
(11.1
)69
(5.1
)156
(11.2
)93
(7.1
)83
(6.1
)69
(5.1
)119
(9.1
)1276
100
CD
8bla
stday
10
23
(1.2
)28
(2.1
)28
(2.1
)15
(1.1
)14
(1.1
)21
(1.2
)27
(2.1
)7
(0.2
)41
(3.1
)14
(1.1
)21
(1.2
)14
(1.1
)47
(3.2
)300
23
1.5
CD
8D
C/D
Cle
uday
10
10
(0.2
)18
(1.1
)18
(1.1
)16
(1.1
)12
(0.2
)21
(1.2
)19
(1.1
)1
(0.1
)18
(1.1
)11
(0.2
)18
(1.1
)11
(0.2
)32
(2.1
)205
16
Pat
ient
#538
CD
4day
0107
(7.2
)108
(7.2
)112
(8.1
)93
(6.2
)109
(7.2
)99
(7.1
)127
(9.1
)89
(6.1
)73
(5.1
)90
(6.1
)125
(9.1
)98
(7.1
)157
(11.1
)1387
100
CD
4bla
stday
10
29
(2.1
)24
(1.2
)33
(2.1
)17
(1.1
)10
(0.2
)21
(1.2
)38
(2.2
)13
(0.2
)47
(3.1
)14
(1.1
)26
(1.2
)13
(0.2
)52
(3.2
)337
24
0.6
CD
4D
C/D
Cle
uday
10
44
(3.1
)21
(1.2
)42
(3.1
)21
(1.2
)31
(2.1
)100
(7.1
)43
(3.1
)22
(1.2
)83
(5.2
)3
(0.1
)40
(2.2
)26
(1.2
)93
(6.2
)569
41
CD
8day
093
(6.2
)97
(6.2
)113
(8.1
)108
(7.2
)126
(9.1
)78
(5.2
)114
(8.1
)118
(8.2
)114
(8.1
)107
(7.2
)114
(8.1
)73
(5.1
)121
(8.2
)1376
100
CD
8bla
stday
10
31
(2.1
)19
(1.1
)36
(2.2
)23
(1.2
)17
(1.1
)24
(1.1
)41
(2.2
)7
(0.2
)50
(3.1
)13
(0.2
)19
(1.1
)20
(1.1
)44
(3.1
)344
25
0.7
CD
8D
C/D
Cle
uday
10
58
(4.1
)39
(2.2
)38
(2.2
)27
(1.2
)23
(1.2
)41
(2.2
)46
(3.1
)18
(1.1
)80
(5.2
)15
(1.1
)41
(2.2
)22
(1.2
)80
(5.2
)528
38
Counti
ng
and
sum
min
gup
all
Jbfa
mil
ypea
ks,
resp
ecti
vel
y,
resu
lted
inth
eto
tal
Jbpea
ksc
ore
sfo
rea
chfr
acti
on.
Acc
ord
ing
toth
eto
tal
Jbpea
ksc
ore
s,th
eper
centa
ges
refe
rrin
gto
the
tota
lpea
knum
ber
are
show
nin
bra
cket
s.T
he
rest
rict
ion
of
the
TR
reper
toir
eaf
ter
bla
stan
dD
Cle
ust
imula
tion
isex
pre
ssed
asra
tio
Clin Exp Med
123
the blast-stimulated T cells (74 vs. 57 %), but also dis-
played a more sustained killing activity (83 vs. 45 %), as
measured after a 3- and 24-h co-incubation with leukemic
target cells (Fig. 2). The importance of cytotoxic CD4?
cells to suppress leukemia colony formation in vitro has
been discussed previously [38, 39] and might be in line
CD4+ T cell fraction in vivo: in vitro:
leu
base pair position →
CD8+ T cell fraction
3 months after SCT 10 days after stimulation with DC/DC
in vivo: in vitro:
leu3 months after SCT 10 days after stimulation with DC/DC
base pair position →
fluo
resc
ence
inte
nsit
y →
fluo
resc
ence
inte
nsit
y →
Fig. 5 Comparison of Vb3 and Vb13.1 T-cell receptor repertoires as
derived from patient samples (#538) 3 months after allogeneic
transplantation (in vivo) and T-cell receptor repertoires as derived
after 10-day in vitro stimulation of the corresponding donor
lymphocytes with DCleu generated from the blasts of the same
patient. The line on the top shows both Vb3 (first Gaussian-like
distribution) and Vb13.1 (second Gaussian-like distribution) families.
The Vb3 family peaks are located between the 160 and 200 base pair
position, whereas the Vb13.1 family exhibits a base pair length of
220–240. The lines below show the corresponding Jb families labeled
with three different colors: The second line presents the Jb families
1.1 (green), 1.2 (blue) and 1.3 (black). In the third line, the Jb families
1.4 (green), 1.5 (blue) and 1.6 (black) are presented. The forth line
involves the Jb families 2.1 (green), 2.2 (blue) and 2.3 (black). In the
fifth line, the Jb families 2.4 (green), 2.5 (blue) and 2.6 (black) are
shown, and in the bottom line, the Jb family 2.7 (green) is presented.
After in vivo and in vitro stimulation identical rearrangements of Vband Jb families with the same CDR3 length could be detected (redcircles). Within the CD8 T-cell fraction, the highlighted circlesdisplay the rearrangement of the Jb family 2.7 with the corresponding
Vb13.1 family, and within the CD4 fraction the Jb family 2.2 with the
corresponding Vb3 family (color figure online)
Clin Exp Med
123
with the observation that CD8? T cells can be depleted
from the DLT without jeopardizing the GvL effect, while
concurrently diminishing GvHD [40]. So far, it is under
investigation whether in vivo CD4? cells additionally have
to recruit leukemia-reactive, minor H-antigen-specific
CD8? cells to exert effective cytolytic activity [41]. In
cases #546 and #538, the induced effector cell population
displayed a more balanced CD4:CD8 ratio resulting in
anergy or even blast growth. But so far, it is still unclear
and remains speculative whether the failure to mediate
protective immunity was due to a relative increase in CD8?
regulatory T cells controlling an outgrowth of antigen-
triggered CD4? T cells or a relative increase in CD4?
regulatory T cells dominating the CD4? T-cell subset [42,
43]. In marked contrast, the stimulatory DC/DCleu fractions
generated in cases #546 and #538 consisted of a lesser
proportion of mature DCs as well as DCleus as compared to
case #502. This was in line with previous observations,
where decreased proportions of these cells were correlated
with a reduced cytotoxicity of the induced effector cell
population [23, 24].
To further delineate these different response patterns,
the T-cell receptor repertoire of the induced CD4? and
CD8? T-cell subsets were analyzed. Thereby, immuno-
scope spectratyping of the Vb gene families revealed that
stimulation with both blast and DC/DCleu caused a signif-
icant clonal restriction of the TR repertoire within both
T-cell subsets, whereas stimulation with DC/DCleu seemed
to have more pronounced effects. Although the CD8?
T-cell compartment per se displayed the highest grade of
restriction, the CD4? T-cell subset seemed to be relatively
more affected by stimulation with DC/DCleu (Tables 3, 4).
Additional analysis of the Jb gene families displayed a
comparable restriction picture. Again, stimulation with
DC/DCleu fractions induced a relatively higher grade of TR
restriction within the CD4? T-cell subset (Table 4; #502
and #546). This was not the case for patient #538. In this
context, DC/DCleu stimulation displayed in both T-cell
subsets a lesser grade of restriction of the Jb gene families.
This might indicate that in case #538, defined Vb gene
segments were rearranged with a higher diversity of Jbgene segments. Although the frequency of the expressed Jbgene families appeared to be very individual, in both T-cell
subsets rearrangements with Jb2.1, Jb2.3 and Jb2.7 seg-
ments seemed to be preferred [44]. So far, no favored Vb/
Jb TR combinations could be established.
To enable comparability of the various TR-Vb profiles,
spectratype data were additionally converted to frequency
distributions and Hamming distance (HD) scores calcu-
lated, thus resulting in a more objective and sensitive data
interpretation. Interestingly, although in patient #538, HD
scores at first glance suggested blast simulation to be
associated with a more restricted profile than DCleu stim-
ulation within the CD8 compartment, as exemplified in
Fig. 4c for the Vb3 and Vb13.1 families, calculation of the
HD scores for all Vb families further confirmed superiority
of DCleu stimulation in line with visual peak evalution.
However technically, immunoscope spectratyping does
not provide any sequence information about the restricted
TR clones subsumed by the displayed peaks. Thus to
demonstrate the occurrence of genetically identical TR bchain clones, the respective clones have to be sequenced.
Thereby in case #538, a CDR3 sequence pattern rearranged
by the Vb13.1 and Jb2.7 genes was found to be identical
within the CD8? T-cell compartment as generated in vitro
after DC/DCleu-mediated stimulation or derived from the
patient material isolated 3 months after haploidentical
transplantation. In vitro, this specific b chain sequence was
found to be tenfold enriched and might indicate the
potential of this approach to predict TR restriction patterns
in vivo. Unfortunately, due to restricted patient material, no
defined antigen specificity of this identified clone could be
established.
Taken together, our results demonstrate that in vitro
stimulation with DC/DCleu fractions result in TR restriction
patterns that might anticipate evolving T-cell repertoires in
vivo. Subsequent analysis via spectratyping in combination
with additional functional assays will further specify the
induced T-cell response pattern. Thus, Vb/Jb T-cell
selection strategies utilizing clonal TR sequence informa-
tion might pave the way for adoptive, leukemia-directed
T-cell therapy or provide the opportunity to anticipate the
risk of relapse at an early stage while monitoring the
defined T-cell clones during a clinical course.
Acknowledgments This work was supported by grants of the
Deutsche Forschungsgesellschaft (SFB/TR 36) to Hans-Jochem Kolb,
Susanne Reuther and Arndt Borkhardt; R. Buhmann was supported by
Fig. 6 Sequencing of the Vb13.1/Jb2.7 recombination was per-
formed for the potential cytotoxic CD8 fraction according to an
identical restriction pattern found 3 months after transplantation and
after in vitro stimulation with DCleu as shown in Fig. 5. The
nucleotide sequences are translated into amino acid sequences
Clin Exp Med
123
grants of the EU (Stemdiagnostics/LSHB-CT-2007037703). We are
indebted to many nurses and physicians for their unconditional
assistance in patient care, referral of patient material and data col-
lection. For extended bioinformatic assistance, we are cordially
indebted to Jens Stoye and Nils Hoffmann. Parts of the results pre-
sented in this manuscript were worked out in the doctoral thesis of
Susanne Reuther.
Conflict of interest None.
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