Immune responses to RHAMM in patients with acute myeloid leukemia after chemotherapy and allogeneic stem cell transplantation.

Abstract

Leukemic blasts overexpress immunogenic antigens, so-called leukemia-associated antigens like the receptor for hyaluronan acid-mediated motility (RHAMM). Persistent RHAMM expression and decreasing CD8+ T-cell responses to RHAMM in the framework of allogeneic stem cell transplantation or chemotherapy alone might indicate the immune escape of leukemia cells. In the present study, we analyzed the expression of RHAMM in 48 patients suffering from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Furthermore, we correlated transcripts with the clinical course of the disease before and after treatment. Real-time quantitative reverse transcriptase polymerase chain reaction was performed from RNA of peripheral blood mononuclear cells. T cell responses against RHAMM were assessed by tetramer staining (flow cytometry) and enzyme-linked immunospot (ELISPOT) assays. Results were correlated with the clinical outcome of patients. The results of the present study showed that almost 60% of the patients were RHAMM positive; specific T-cells recognizing RHAMM could be detected, but they were nonfunctional in terms of interferon gamma or granzyme B release as demonstrated by ELISPOT assays. Immunotherapies like peptide vaccination or adoptive transfer of RHAMM-specific T cells might improve the immune response and the outcome of AML/MDS patients.

Full text

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Hindawi Publishing Corporation
Clinical and Developmental Immunology
Volume 2012, Article ID 146463, 9 pages
doi:10.1155/2012/146463
Research Article
ImmuneResponsesto RHAMM inPatients with
AcuteMyeloidLeukemiaafterChemotherapyand Allogeneic
StemCellTransplantation
R.Casalegno-Gardu˜ no,1,2C.Meier,3A.Schmitt,1,2A.Spitschak,3I.Hilgendorf,1
S.Rohde,1C.Hirt,4M.Freund,1B. M.P¨ utzer,3andM. Schmitt2
1Department of Internal Medicine III, University of Rostock, 18057 Rostock, Germany
2Cellular Immunotherapy, Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany
3Department of Vectorology and Experimental Gene Therapy, Biomedical Research Center (BMFZ),
University of Rostock, 18057 Rostock, Germany
4Department of Internal Medicine C, Hematology and Oncology, University of Greifswald, 17487 Greifswald, Germany
Correspondence should be addressed to M. Schmitt, michael.schmitt@med.uni-heidelberg.de
Received 30 January 2012; Revised 29 March 2012; Accepted 30 March 2012
Academic Editor: Masoud H. Manjili
Copyright © 2012 R. Casalegno-Gardu˜ no et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Leukemic blasts overexpress immunogenic antigens, so-called leukemia-associated antigens like the receptor for hyaluronan acid-
mediatedmotility(RHAMM).PersistentRHAMMexpressionanddecreasingCD8+T-cellresponsestoRHAMMintheframework
of allogeneic stem cell transplantation or chemotherapy alone might indicate the immune escape of leukemia cells. In the present
study, we analyzed the expression of RHAMM in 48 patients suffering from acute myeloid leukemia (AML) and myelodysplastic
syndrome (MDS). Furthermore, we correlated transcripts with the clinical course of the disease before and after treatment. Real-
timequantitativereversetranscriptasepolymerasechainreactionwasperformedfromRNAofperipheralbloodmononuclearcells.
TcellresponsesagainstRHAMMwereassessedbytetramerstaining(flowcytometry)andenzyme-linkedimmunospot(ELISPOT)
assays. Results were correlated with the clinical outcome of patients. The results of the present study showed that almost 60% of
the patients were RHAMM positive; specific T-cells recognizing RHAMM could be detected, but they were nonfunctional in terms
of interferon gamma or granzyme B release as demonstrated by ELISPOT assays. Immunotherapies like peptide vaccination or
adoptive transfer of RHAMM-specific T cells might improve the immune response and the outcome of AML/MDS patients.
1.Introduction
Approximately 80% of patients with acute myeloid leukemia
(AML)reachacompleteremission (CR)afterchemotherapy.
However, half of the patients in CR relapse and only
25% of all AML patients survive more than five years.
Therefore, there is a fervent need for novel therapies to
treat leukemia including immunotherapeutic approaches.
Leukemic blasts overexpress proteins that play an important
role in survival and proliferation of the cells. These proteins
have been designated leukemia-associated antigens (LAAs).
LAAs comprise a broad group of proteins including the
receptor for hyaluronic-acid-mediated motility (RHAMM)
[1]. CD8+T-cell responses against RHAMM have been
identified in AML patients [2]. Nevertheless, it remains to
be elucidated why immunogenic LAAs are expressed but not
sufficiently recognized, and why LAA+malignant cells are
not subjected to lysis through specific CD8+T cells. Immune
escape might become effective through downregulation of
LAAs or hampering of the proper function of T cells [3].
Only limited information is available on the expression of
LAAs before and after chemotherapy and/or allogeneic stem
cell transplantation (allo-SCT). Therefore we investigated
here the expression of RHAMM as well as the frequency
of RHAMM-specific CD8+T lymphocytes before and after
chemotherapy/allo-SCT. As a future perspective, rather poor

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2Clinical and Developmental Immunology
immune responses to RHAMM might be enhanced through
immunotherapeutic approaches such as peptide vaccination
and adoptive transfer of specific T-cell responses in the
context of chemotherapy and allo-SCT [1].
2.MaterialandMethods
2.1. Blood Samples from Patients and Healthy Donors. We
collected 173 peripheral blood samples from 48 patients
suffering from AML/MDS after obtaining the patients’
informed consent. This study was approved by the local
ethical committee. Serial peripheral blood samples were
collected at diagnosis, after chemotherapy and/or after allo-
SCTwithimmunosuppression,inCRorduringmaintenance
therapy at sequential time intervals during follow-up and
at relapse. Both peripheral blood mononuclear cell (PBMC)
and bone marrow mononuclear cell (BMMC) samples were
prepared using Ficoll Biocoll separating solution (Biochrom,
Berlin, Germany) and cryopreserved according to standard
protocols. PBMC samples were obtained from 10 healthy
donors and used as negative controls.
2.2. Real-Time Quantitative Reverse Transcriptase Polymerase
Chain Reaction (RQ-RT-PCR). RNA was isolated from a
minimum of 2×106cells using RNeasy plus minikit (QIA-
GEN, D¨ usseldorf, Germany). Five hundred nanograms of
RNA were reversely transcribed into cDNA using the iScript
cDNAsynthesiskit(BioRad,Munich,Germany).Thereverse
transcription (RT) products were diluted with 70μL of
molecular-biology water (Sigma-Aldrich). Nine micro liters
wasusedperwell.Primers/probes(TaqManGeneExpression
Assays, Invitrogen) were diluted in TaqMan 2xPCR Master
Mix according to manufacturer’s instructions. Standard
curves for RHAMM and ABL were established for each
run of RT-PCR using four dilution steps per gene. Copy
numbers were calculated by http://www.endmemo.com/.
Reactions were tested in duplicate using the ABI PRISM
7900 sequence detection system (Applied Biosystems) and
standard conditions with 40 cycles of amplification in 20μL
of volume.
2.3. Mixed Lymphocyte Peptide Culture (MLPC). MLPC was
performed as described elsewhere [4]. Briefly, specific CD8+
T cells were selected from PBMCs and BMMCs by magnetic-
activated cell sorting (MACS) columns (Miltenyi). CD8−
fraction was irradiated with 30Gy and loaded with test
or control peptides (20μg/mL) or cultured with medium
alone (no peptide). Peptide sequences of RHAMM and
control peptides derived from phosphoprotein-65 of the
cytomegalovirus (CMVpp65) and influenza matrix protein
(IMP) were ILSLELMKL, NLVPMVATV, and GILGFVFTL,
respectively. CD8+and CD8−cells were cocultured in a ratio
of 1:4. MLPC was supplemented with 10U/mL IL-2 (Sigma
Aldrich) and 20ng/mL IL-7 (Miltenyi) on day 1. Cytotoxic
T lymphocytes (CTLs) were harvested on day seven for
enzyme-linked immunospot (ELISPOT) assay and/or flow
cytometry analysis when sufficient numbers of CD8+cells
were collected.
2.4. Mini-MLPC. The MLPC approach was modified into
a mini-MLPC in case that insufficient numbers of CD8+
cells were obtained from MACS separation. Mini-MLPCs
wereperformedinround-bottom96-wellmicrotiterplatesin
RPMI-1640 culture medium supplemented with 10% heat-
inactivated human AB serum, 10U/mL IL-2, and 20ng/mL
IL-7. The ratio was maintained as in the MLPC (1×104
CD8+and 4×104CD8−cells, 1:4). Number of cells per
well was based on the work by Distler et al. [5]. Proliferation
observed in mini-MLPCs was comparable to proliferation in
conventional MLPCs.
2.5. ELISPOT for Interferon Gamma (IFN-γ) and Granzyme
B. IFN-γ and granzyme B ELISPOT assays were performed
as described elsewhere [4] to determine specific recognition
of RHAMM peptide-positive target cells according to manu-
facturer’s instructions (BD, San Diego, USA).
2.6. Flow Cytometry Analysis. The frequency of RHAMM-
specific T cells was determined by flow cytometry. Tetramer
staining was performed as described previously [2]. Briefly,
lymphocytes were stained with RHAMM-specific tetramers
and subsequently with conjugated antibodies to CD3 and
CD8 (BD Biosciences). Fluorescein isothiocyanate (FITC),
peridinin-chlorophyll protein (PerCP), and phycoerythrin
(PE) were used as fluorochromes. A minimum of 2×104
cells were acquired. Flow cytometry was performed on a
Calibur cytometer (BD Biosciences). Appropriated isotype
controls were included in each experiment. Data were
analyzed using the flow cytometry analysis software FlowJo
(Tree Star, Inc, USA). The frequency of tetramer CD8+T
cells was considered positive if it was 2-fold or higher than
the frequency of CD8+cells counterstained with a tetramer
recognizing an irrelevant peptide.
2.7. Clinical Status of the Patients. The clinical status of
patients was obtained from the clinical data base of the
Department of Internal Medicine III, University of Rostock.
Clinical features of the patients such as chimerism analysis,
cytogenetics, HLA, CMV status, and therapy were evaluated
by the Department of Internal Medicine III, University of
Rostock. The FLT3 status was assessed at the Department
of Hematology/Oncology at the University of Greifswald,
Greifswald, Germany. AML cases were classified according to
the FAB criteria and characterized at the cytogenetic level.
CR was defined according to standard criteria.
2.8. Statistical Analysis. Statistical analyses were performed
using Stat Graphics Plus 5. The standard Wilcoxon signed-
ranktestwasusedfornonparametriccomparisonsofmedian
expression of RHAMM before and after treatment, as the
data were paired and not normally distributed. Mann-
Whitney U-test was used for nonparametric comparisons
of median expression of RHAMM in healthy donors and
patients. Statistical significance was considered if the P value
was <0.05.

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Clinical and Developmental Immunology3
3.Results
3.1. Patients’ Characteristics. We screened 48 AML/MDS
patients in a prospective study. Twenty-one patients received
allo-SCT, whereas 27 received only chemotherapy under
conventional protocols. Our cohort of patients maintained
a ratio of almost 1:1 between male (n = 23) and female
patients (n
= 25). A normal karyotype was found in
19 patients, and aberrant karyotype in 17 patients, and
a complex karyotype in seven patients. The karyotype of
five patients was not accessible. There was no significant
difference between the age of men and women (P = 0.5)
at the time of diagnosis. The mean follow-up time of the
patients was 272 days (median: 225 days). Nine patients
died from leukemia, and three patients from diseases not
related to leukemia, that is, encephalitis, pneumonia, and
graft versus host disease.
3.2. Expression of RHAMM Transcripts before and after Treat-
ment. Expression of RHAMM in the peripheral blood of
healthy donors (n
= 10) was very low (median: 318;
range 97–730 RHAMM copies/104ABL copies). In contrast
RHAMM transcripts were significantly higher in patients
before treatment (median: 768; range: 184–36, 160; P =
0.001). In order to compare different treatments, patients at
diagnosis were split in two groups: patients after chemother-
apy alone and patients after allo-SCT (Figure 1). Peripheral
blood was collected from 22 AML patients at the time
of diagnosis. Thirteen patients were RHAMM positive
(59%), whereas nine were negative (41%). The expression
of RHAMM was considered positive when it was higher
than the highest value measured in the peripheral blood
of healthy donors. There was no significant difference in
expression of RHAMM in the peripheral blood of patients
before treatment according to FLT3-ITD status (positive
versus negative, P = 0.89), gender (P = 0.66), or karyotype
(normal versus aberrant, P = 0.29; normal versus complex,
P = 0.75; aberrant versus complex, P = 0.40).
Furthermore, we aimed to determine the expression
of RHAMM before and after chemotherapy alone or allo-
SCT. Therefore we measured absolute copy numbers of
this gene in AML/MDS-diagnosed patients. There was no
significant difference before and after treatment, neither by
chemotherapy (P = 0.83) nor by allo-SCT (P = 0.28).
However, we observed higher transcript numbers during
CR of patients that received allo-SCT when compared to
thosewhoreceivedchemotherapy(P = 0.009).Furthermore,
RHAMM was also equally expressed before and after allo-
SCT in a cohort of patients in CR. This group also showed
highercopynumbersofRHAMM aftertransplantationwhen
compared to the group that was treated with chemotherapy
(P = 0.007, Figure 1).
3.3. RHAMM-Specific CTLs in Healthy Donors. RHAMM-
specific T cells were observed in three of ten healthy
donors, that is, in HD 155, HD 663, and HD 005, at low
frequencies (0.11%, 0.33%, and 0.12%, resp.) with cells
in the CD3+CD8+gate set 100% (Figure 2(g) with controls
CR DxHD
Chemotherapy
CRDx cCR CR
Allo-SCT
2,000
3,000
1,000
4,000
5,000
0
P = 0.002
P = 0.008
P = 0.009
P = 0.83
P = 0.007
P = 0.28
P = 0.15
Normalized RHAMM expression
(RHAMM/ABL∗10000)
Figure 1: RHAMM expression before and after chemotherapy
alone or allo-SCT. Patients at diagnosis had higher copy numbers
when compared to healthy donors (chemotherapy group, P =
0.002; allo-SCT group, P = 0.008). There was no significant differ-
ence in expression of RHAMM before (white boxes) and after (grey
boxes) treatment, neither for chemotherapy alone (P = 0.83) nor
for allo-SCT (P = 0.28), in patients at diagnosis that reached a CR
after treatment. Also patients in CR that received allo-SCT showed
no difference in RHAMM transcripts before (white boxes) and after
(grey boxes) transplant (P = 0.15). However, patients that received
allo-SCT had higher copy numbers when compared to those who
received chemotherapy during the CR (P = 0.009) or continuous
CR (cCR, P = 0.007). Medians are shown in the box plots.
RHAMM: receptor for hyaluronic acid mediated motility, allo-SCT:
allogeneic stem cell transplantation, CR: complete remission, P-
value: probability value.
Figures 2(c)–2(f)). Moreover, an activity of these RHAMM-
specific CTLs was detected in two healthy donors (HD 155
and 669) as for IFN-γ secretion evaluated by ELISPOT
assays (Figure 2(a)). CTLs from healthy donor 669 were not
sufficient in number to perform flow cytometry analysis.
3.4. RHAMM-Specific CTLs in Patients. Longitudinal studies
of RHAMM-specific CD8+T cells were only possible with
samples of ten patients who overexpressed RHAMM (29/48
patients) and were HLA-A2 positive, as all peptides used
in this study were HLA-A2 restricted. The frequency of
RHAMM-specific CD8+T cells in the peripheral blood was
measuredbyflowcytometryduringthecourseofthedisease.
Furthermore their activation status and potential to kill
RHAMM+malignant cells was assessed by secretion of IFN-
γ and granzyme B, respectively. As displayed in Figure 3(c),
RHAMM-specific CTLs were detected at a frequency of
1.24% up to 5.62% of all CD8+T cells, and 0.03% up to
1.14% of all cells in the lymphocyte gate. In ELISPOT assays
a general activity of CD8+T cells (Figures 3(a) and 3(b))
was detected at the time of diagnosis (Dx) and in CR. Thirty
days prior to allo-SCT a stronger release of granzyme B by
RHAMM-R3-stimulated CD8+T cells than by unstimulated
T cells was detected. After allo-SCT a general silencing of
T cells as for release of both IFN-γ and granzyme B was
detectable (Figures 3(a) and 3(b)). In accordance with these
findings flow cytometry analysis revealed that the number of
RHAMM-tetramer+CD8+T cells vanished over the time.

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4Clinical and Developmental Immunology
Spots/10,000 CD8+T cells
0
100
200
300
400
500
600
700
HD 005 HD 155 HD 410 HD 900 HD 676 HD 669 HD 663
No peptide
RHAMM
IMP
CMV
IFN-γ
(a)
No peptide
RHAMM
IMP
CMV
Spots/10,000 CD8+T cells
0
100
200
300
400
500
600
700
HD 005 HD 155 HD 410 HD 900 HD 676 HD 669 HD 663
Granzyme B
(b)
100
100
101
101
102
102
FL 2
103
103
104
104
FL 1
R3
0%
0%
(c)
100
101
102
103
104
100
101
102
103
104
CD8
G250 tetramer
R3
0%
0%
(d)
CD8
100
100
101
101
102
102
103
103
104
104
CMV tetramer
R3
13%
3.82%
(e)
CD8
IMP tetramer
100
100
101
101
102
102
103
103
104
104
R3
3.36%
1.21%
(f)
CD8
RHAMM tetramer
100
100
101
101
102
102
103
103
104
104
R3
0.11%
0.04%
(g)
Figure 2: RHAMM-specific CD8+T-cell frequencies in healthy donors. Cells were stimulated in an MLPC for 7 days with different peptides
and tested for their reactivity in ELISPOT assays for IFN-γ (a) and granzyme B (b) release. Stimulation of cells without any peptide was used
as negative control (no peptide), whereas T cells stimulated with CMV and IMP peptides served as positive controls. RHAMM-specific T
cells could be detected in two healthy donors (HD 155 and HD 669) by IFN-γ ELISPOT, (c)–(g) RHAMM-specific T-cell frequencies were
determined by flow cytometry in HD 155. Reported frequencies correspond to gated CD3+CD8+T cells (upper numbers), and from all cells
in the lymphocyte gate (lower numbers). (c) Fluorescence minus one (FMO) was used as negative control to assess the intrinsic fluorescence
of the cells. (d) As a further negative control, cells were cultured in the absence of any peptide and stained with tetramers specific for the
irrelevant antigen G250. As positive controls, CD8+T cells were stimulated with either (e) CMVpp65 peptide or (f) IMP-derived peptide,
(g) CD8+T cells were stimulated with RHAMM peptide, (e)–(g) CD8+T cells were stained with respective tetramers.
In another AML patient (Figure 4), RHAMM-specific
CD8+T cells were detected by flow cytometry which cer-
tainly constituted a distinct subpopulation of RHAMM-
specific CD8+T cells (Figures 4(f) and 4(j)), as proven by
a number of negative controls (Figures 4(c)–4(e) and 4(g)–
4(i)) including RHAMM-tetramer stained CD8+cells which
were not stimulated by any peptide (Figures 4(e) and 4(i)).
InterestinglyweobservedageneralactivationofCD8+Tcells

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Clinical and Developmental Immunology5
0
−98
−30
−9+69 +103 +206 +221 +279
Allo-SCT
IFN-γ
Spots/10,000 CD8+T cells
(days)
No peptide
RHAMM
CMV
Relapse CRDx
120
100
80
140
20
40
60
(a)
100
Spots/10,000 CD8+T cells
−98
−30
−9
+206 +221 +279
Granzyme B
20
40
60
80
0
RelapseCRDx
Allo-SCT
(days)
No peptide
RHAMM
CMV
(b)
CD8
RHAMM tetramer
2.11%
0.04%
100
100
101
101
102
102
103
103
104
104
R3
+206days
2.61%
1.41%
100
100
101
101
102
102
103
103
104
104
R3
−9days
5.62%
1.14%
100
100
101
101
102
102
103
103
104
104
R3
−98days
2.91%
3 1.2 %
100
100
101
101
102
102
103
103
104
104
R3
−30days
0.03%
1.24%
100
100
101
101
102
102
103
103
104
104
R3
+221days
(c)
Figure 3: Longitudinal study of RHAMM-specific CTLs in a patient with AML that received allo-SCT. PBMCs were collected from an AML
patient at different time points before or after allo-SCT as indicated and subjected to MLPC. CD8+T cells showed RHAMM-specific release
of IFN-γ (a) and granzyme B (b) at one time point (30 days prior to transplant) when the patient was in CR. This active T-cell population
was lost after allo-SCT and not reconstituted when the patient relapsed. (c) CD8+T cells were stimulated with RHAMM peptide and stained
withrespective tetramer. Frequencies of RHAMM-specificCTLs vanished over the time, as detected by flow cytometry. Reported frequencies
correspond to the gate of CD3+CD8+T cells (upper numbers) and to all cells in the lymphocyte gate (lower numbers).
at the time of relapse of the patient with no difference of
smoldering and progressive disease(Figures 4(a) and 4(b)).
This might be due to high concentrations of RHAMM
on proliferating malignant cells stimulating specific T-cell
responses.
In a third patient with AML antigen-specific CD8+T
cells were detected in both peripheral blood (PB) and bone
marrow (BM) to release IFN-γ and granzyme B in ELISPOT
assays (Figure 5). RHAMM-specific CD8+T cells were able
to release IFN-γ and granzyme B when the patient relapsed