From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Blood First Edition Paper, prepublished online May 14, 2009; DOI 10.1182/blood-2009-03-213256
Functionally Active Virus-Specific T-Cells that Target CMV, Adenovirus and EBV
can be Expanded from Naïve T-cell Populations in Cord Blood and will Target a
Range of Viral Epitopes
Patrick J. Hanley1,3, Conrad Russell Young Cruz1, Barbara Savoldo1,2, Ann M Leen1,2,
Maja Stanojevic1, Mariam Khalil1, William Decker6, Jeffrey J Molldrem6, Hao Liu1,4,
Adrian P Gee1,2, Cliona M Rooney1,2,5, Helen E Heslop1,2,3, Gianpietro Dotti1,3,4, Malcolm
K. Brenner1,2,3, Elizabeth J Shpall6, Catherine M. Bollard1,2,3,4
1
Center for Cell and Gene Therapy, Departments of
4
Medicine, 5Virology, Baylor College of Medicine, Texas Children’s Hospital and The
2
Pediatrics,
3
Immunology,
Methodist Hospital, Houston, Texas and 6Department of Stem Cell Transplantation and
Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, Texas
Corresponding author:
Catherine Bollard, MD
Center for Cell and Gene Therapy
Baylor College of Medicine
6621 Fannin Street, MC 3-3320
Houston, Tx 77030USA
Phone:
832 824 4781
Fax:
832 825 4668
Email: [email protected]
Short Title for running head: Virus-specific Cytotoxic T-cells from Cord Blood
Copyright © 2009 American Society of Hematology
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ABSTRACT
The naive phenotype of cord blood (CB) T-cells may reduce graft versus host disease
after umbilical cord blood transplant (CBT), but this naivety and their low absolute
numbers also delays immune reconstitution, producing higher infection-related mortality
that is predominantly related to CMV, adenovirus (Adv) and EBV. Adoptive
immunotherapy with peripheral blood-derived virus-specific cytotoxic T-lymphocytes
(CTLs) can effectively prevent viral disease after conventional stem cell transplantation,
and we now describe the generation of single cultures of CTLs from CB that are specific
for multiple viruses. Using EBV-infected B-cells transduced with a clinical grade
Ad5f35CMVpp65 adenoviral vector as sources of EBV, Adv and CMV antigens, we
expanded virus-specific T-cells even from CB T-cells with a naive phenotype. After
expansion, each CTL culture contained both CD8+ and CD4+ T-cell subsets,
predominantly of effector memory phenotype. Each CTL culture also had HLA restricted
virus-specific cytotoxic effector function against EBV, CMV and Adv targets. The CB
CTLs recognized multiple viral epitopes, including CD4-restricted Advhexon epitopes
and immunosubdominant CD4 and CD8-restricted CMVpp65 epitopes. Notwithstanding
their naive phenotype, it is therefore possible to generate trivirus-specific CTLs in a
single culture of CB, which may be of value to prevent or treat viral disease in CBT
recipients. This study is registered at www.clinicaltrials.gov as NCT00078533.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
INTRODUCTION
Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for
selected patients with high-risk hematologic malignancies. However a significant
proportion of patients – especially non-Caucasians - lack a marrow or peripheral blood
stem cell donor. Umbilical cord blood (CB) has therefore emerged as an important
alternative source of stem cells for allotransplant patients.1;2
The major advantages of CB transplants (CBT) compared to unrelated adult donor stem
cells include more rapid procurement of the graft, the requirement for less stringent HLA
matching, the higher likelihood of finding a match for ethnic minorities, and a decreased
incidence of graft versus host disease (GVHD). Although a major disadvantage of CB is
the low cell stem cell dose, which results in delayed engraftment, there is also delayed
immune recovery, particularly in mismatched unrelated cord blood transplants utilizing
anti-thymocyte globulin (ATG).3 This is attributed to the small numbers of T-cells
transferred, the absence of memory T-cells within the CB grafts and the apparent
hyporesponsiveness of CB antigen presenting cells. Consequently, CB recipients are
susceptible to an array of viral and other infections that are the leading cause of death in
these patients. 3-7
Cytomegalovirus (CMV), Epstein Barr Virus (EBV) and Adenovirus (Adv) are particularly
problematic in the immunocompromised recipient, and are associated with significant
morbidity and mortality.8-10 Although anti-viral drugs can be beneficial for CMV and EBV,
effective agents are unavailable for adenovirus, all have substantial toxicities and drug
resistance may occur. These deficiencies in conventional therapeutics have increased
interest in an immunotherapeutic approach to viral disorders, and adoptive transfer of
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
monoculture-derived multi-virus specific CTL from CMV-seropositive donors can prevent
and treat CMV, EBV and Adenoviral disease post-HSCT without significant toxicities.11
There are significant conceptual obstacles to extending this approach to the recipients of
CB transplants. Only limited numbers of CB T-cells are available for manipulation, and
these cells have a naïve phenotype, making them incapable of conferring protection
against CMV, EBV and Adv during engraftment.3;12 Hence, the development of virusprotective T-cell therapy for CBT recipients requires the priming and extensive
expansion of naïve T-cells rather than the more limited and simple direct expansion of a
pre-existing virus specific memory T-cell population. This task is made even more
challenging since CB T-cells have lower cytotoxic activity and higher activation-induced
cell death than peripheral blood-derived T-cells.13;14 These limitations have prevented
the successful generation of virus-specific cord blood-derived CTL in sufficient numbers
for clinical use as prophylactic therapy of CMV, EBV and adenoviral infections after
CBT.
It has become evident that in addition to antigen presentation and intercellular
costimulation, the ex vivo priming of antigen-specific T-cells requires the presence of
appropriate soluble cytokines. Hence, IL-7, IL-15, and IL-12 respectively decrease the
antigen concentration threshold, direct T-cells towards a central memory phenotype, and
influence the polarization of Th1 cells. In combination these cytokines augment the
generation of antigen specific CTL from naïve T-cells. 15
We now describe how Ad5f35pp65-transduced CB-derived antigen presenting cells can
be used to generate large numbers of T-cells that are specific for both CMV and
Adenovirus even from the naive T-cell population in human CB. Incorporation of EBVtransformed B-lymphoblastoid cell lines in the antigen presenting cells further allows the
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Adv/CMV specificity of the CB T-cells to be extended to EBV. The cells we describe
have broad epitope specificity, and are cytolytic to CMV, Adv and EBV target cells.
Hence they may be protective in vivo.
MATERIALS AND METHODS
Cord Blood Units
Virus-specific CTL were generated from CB units obtained frozen from the MDACC Cord
Blood bank or fresh from mothers who had consented to the protocol approved by the
IRB of Baylor College of Medicine. To ensure the future clinical feasibility of this
approach, we also froze the fresh CB units in DMSO containing 50% human serum prior
to use for the generation of CB-derived CTL, thereby mimicking the likely clinical setting.
All cord bloods were typed by the HLA laboratory of The Methodist Hospital, and we
used CB from donors with multiple HLA types (Table 1). To further ensure that the
approach would be feasible clinically, a total of only forty million CB mononuclear cells
(which can be obtained from the 20% fraction of frozen CB units) were thawed and used
in the manufacturing process. According to our IRB approval clinical protocol patients
must have a single CB unit matched with the patient at 4, 5, or 6/6 HLA class I
(serological) and II (molecular) antigens.
The unit must be cryopreserved in two
fractions, with a minimum of 2.5x107 total nucleated cells per kg pre-thaw in the fraction
which will be used for the primary transplant. The remaining fraction will be used to
generate the CTLs to give at day 30 or beyond as described below. This cell dose has
been found to support acceptable engraftment in both pediatric and adult patients and is
a commonly used minimal cell dose target in the cord blood transplant community. 16
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Generation of Dendritic Cells (DC) from Cord Blood
Umbilical cord blood was thawed and then purified by Ficoll (Lymphoprep; Nycomed,
Oslo, Norway) gradient separation. 30 x106 CBMC were washed twice and resuspended
in CellGenix media (CellGenix USA, Antioch, IL) and plated at 5x106 cells per well in DC
medium (CellGenix media plus 2 mM L-glutamine) (GlutaMAX, Invitrogen, Carlsbad, CA)
in a 6-well plate (Costar, Corning, NY) for 2 hr at 37°C in a humidified CO2 incubator.
Non-adherent cells were removed by rinsing with 1X PBS (GibcoBRL, Gaithersburg,
Maryland), and refrozen. Loosely adherent cells were cultured in DC media with 800
U/ml GM-CSF (Sargramostim Leukine; Immunex, Seattle, WA) and 500 U/ml IL-4 (R&D
Systems, Minneapolis, MN, USA) for 7 days. IL-4 and GM-CSF were again added on
day 3. On day 5, cells were harvested for transduction.
Transduction of Dendritic cells
As our source of CMV and adenovirus antigens, we used a clinical-grade recombinant
adenovirus type 5 vector pseudotyped with an Adv type 35 fiber, that encoded
CMVpp65.11
On day 5, the cord blood derived dendritic cells were transduced with the clinical grade
Ad5f35CMVpp65 vector11 at a multiplicity of infection (MOI) of 10 i.u. per cell for 2 hours
and matured in a cytokine cocktail of GM-CSF, IL-4, IL-1β, TNF-α, IL-6, (R&D Systems,
Minneapolis, MN) and PGE2 (Sigma, St. Louis, MO) for 2 days. On day 7, the DCs were
harvested, irradiated (30Gy) and then used to stimulate virus-specific CTLs.
Generation and Transduction of EBV-transformed B cell lines from Umbilical Cord
Blood
As the source of EBV antigens, 5 x 106 cord blood mononuclear cells (CBMC) were used
for the establishment of an EBV transformed lymphoblastoid cell line (EBV-LCL), by
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
infection of CBMC with concentrated clinical grade supernatants from a B95-8 working
cell bank.17
Two days before CTL stimulation, LCL were harvested, pelleted and incubated with
Ad5f35CMVpp65 at a MOI of 100 i.u./cell for 90mins at 37°C. The cells were then
resuspended at 5 x 105 cells/ml of complete media (RPMI (Hyclone) plus human serum
and glutamax) and transferred to a 24 well plate at 2mls per well and cultured for 2 days
before use as stimulator cells.
Generation of Multivirus-specific Cultures Derived from Umbilical Cord Blood
Frozen CBMC were thawed, washed and resuspended in 45% RPMI (Hyclone) and 45%
CLICKS (Irvine Scientific) with 10% Human Serum plus GlutaMAXTM-I (CTL medium).
Cells were resuspended at 2x106/ml and co-cultured with autologous, irradiated
transduced DCs at a ratio of 20 CBMC to 1 DC in the presence of the cytokines IL-7, IL12, (R&D Systems) and IL-15 (CellGenix), all at 10ng/mL. Cultures were restimulated on
day 10 with irradiated Ad5f35CMVpp65-transduced LCL at a responder-to-stimulator
ratio of 4:1 plus IL-15 (10ng/ml), and 1 week later with irradiated autologous
Ad5f35CMVpp65-transduced LCL at a responder-to-stimulator ratio of 4:1. IL-2 (50-100
U/ml, Proleukin; Chiron, Emeryville, CA) was added 3 days after the second stimulation
and then twice weekly.
To define the origin of the virus-specific T-cell population we sorted CD45RA/CCR7
double positive (DP) and double negative (DN) CD3+ T-cell populations using flow
cytometry and stimulated the DP and DN populations with transduced DC followed by
transduced LCL as described above.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Peripheral blood derived CTL lines
CTL lines from peripheral blood were prepared from stem cell donors who gave informed
consent, in accordance with the Declaration of Helsinki, upon enrollment in our clinical
trial of virus-specific T cells for the treatment of viral infection after transplant.11 All
protocols were approved by the Baylor College of Medicine institutional review boards
and the National Marrow Donor Program. For the purposes of this analysis, we have
characterized 11 of these CTL lines.
Immunophenotyping
CTL lines were analyzed with monoclonal antibodies to: CD19, CD4, CD8, CD56/16,
TCRαβ, TCRγδ, CD45RA, CD3/28, CD62L, CD4/25, CCR7 (Becton Dickinson).
Samples were acquired on a FACScan flow cytometer (Becton Dickinson) and the data
analyzed using CellQuest software (Becton Dickinson).
Enzyme-Linked Immunospot (ELISPOT) assay
ELISPOT analysis was used to determine the frequency and function of T-cells secreting
IFN-γ in response to pepmixes™ (Jerini, Berlin, Germany) for CMVpp65, AdvHexon,
AdvPenton and CMVIE1 which contain all 15mer peptides of each viral antigen in one
pool.11 ELISPOT assays were performed on the CTL lines. In addition, to define the CD4
and CD8 restricted CMVpp65-specific activity we pulsed CD8+ or CD4+ T-cells (sorted
from CTL lines using flow cytometry) with CMV peptides.18 CBMC stimulated with
CMVIE1 pepmix and Staphylococcal Enterotoxin B (1μg/ml) (Sigma-Aldrich Corporation,
St Louis, MO) served as controls. Spot-forming cells (SFC) were enumerated by Zellnet
Consulting (New York, New York) and compared with input cell numbers to obtain the
frequency of virus reactive T-cells.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Multimers and peptides
To detect CMVpp65-specific T-cells in the CTL lines, we used the soluble HLA-peptide
HLA-A*0201-MLN tetramer prepared by the Baylor College of Medicine Tetramer Core
Facility. Peptides were synthesized by the Baylor College of Medicine Protein Core
Facility or by Proimmune Inc (Springfield, Virginia) .Tetramer staining of CTLs (5 x 105)
is previously described.19 For each sample, 100,000 cells were analyzed as described
above.
Panels of 20mer peptides (overlapping by 15 amino acids) covering the entire amino
acid sequence of CMVpp65 from the AD169 strain and adenovirus hexon from serotype
5 were synthesized.18;20 For CMVpp65, twenty-two peptide pools comprising 2 to twelve
15mer peptides were prepared, so that each 20mer peptide was represented in two
pools.
20
For Advhexon, eleven peptide pools contained 17 or 18 peptides, so that each
20mer peptide was represented in one pool, and we then screened the positive pools as
previously described.18 These CMVpp65 and Advhexon peptide libraries were designed
to identify all possible HLA class I restricted epitopes, which have a length of 9-11 amino
acids, and to also identify HLA class II restricted epitopes, which have lengths of 13 to
17 amino acids.21
Cytotoxicity assay
CTLs were tested for specific cytotoxicity against autologous LCL22, PHA blasts pulsed
with CMVpp65 pepmix (Jerini) or adenovirus hexon pepmix. As control target cells we
used unpulsed PHA blasts, PHA blasts pulsed with irrelevant peptides and HLA class Imismatched LCLs.
51
Cr-labeled target cells were mixed with effector cells at doubling
dilutions to produce the effector: target (E: T) ratios specified. Target cells incubated in
complete medium or 5% Triton X-100 (Sigma) were used to determine spontaneous and
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
maximal
51
Cr release, respectively. After 4 hours (LCLs and PHA blasts) or 6 hours
(fibroblasts), supernatants were collected and radioactivity was measured on a gamma
counter. The mean percentage of specific lysis of triplicate wells was calculated as 100 ×
(experimental release - spontaneous release)/(maximal release - spontaneous release).
Statistical analysis
The Student t test was used to test for significance in each set of values, assuming
equal variance. Mean values ± SD are given unless otherwise stated.
RESULTS
Expanded Cord Blood-derived CTLs are Virus-specific and Polyclonal
We prepared T-cell lines from 9 different cord blood units. After growth on
Ad5f35CMVpp65 transduced dendritic cells and LCLs in the presence of IL-2, IL-7 and
IL-15, we harvested a median of 85.5x106 T-cells (range 60x106 to 2.65x108) by day 21
of culture. All of these cell numbers would be sufficient for current T-cell adoptive
transfer protocols, where 1x107/m2 effectively reconstitute immunity to CMV, EBV and
adenovirus11 and for product safety, function and identity testing (Figure 1a). These
cells were 78.7% CD8+ (range 46-94%) and 31% CD4+ (range 12-54%) (Figure 1b)
Flow cytometric analysis of memory markers revealed a predominance of CD45RO+
CD62L- T-cells (mean 87.9±5%) with a smaller population of CD45RA- CD62L+ T-cells
(mean 14.6±13%). (Figure 1b)
We next used cytotoxicity and IFNγ ELISPOT assays to discover if the T-cell lines were
indeed trivirus specific cytotoxic T lymphocytes (CTLs). Cytotoxic activity was tested
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
against a panel of
51
Cr-labeled autologous and allogeneic target cells and showed that
the CTL lines killed PHA blasts only if they were pulsed with CMVpp65 pepmix or
Advhexon pepmix (40% and 13% respectively at an E: T ratio of 20:1 (Figure 1c)).
Furthermore, autologous EBV-LCLs were also killed in this assay, showing an EBVspecific T-cell component. There was no cytotoxic activity against unpulsed PHA blasts,
PHA blasts pulsed with CMVIE1 pepmix (Figure 1c) or against HLA-mismatched EBVLCLs (data not shown).
Similarly, Figure 1d illustrates that after three stimulations, virus-specific T-cell lines
(n=9) can secrete IFNγ in response to EBV, CMVpp65- and Adv hexon-expressing
target cells with a mean and SD of 157 +/- 134, 209 +/- 210 and 74+/- 65 spot-forming
cells/1x105
T-cells,
following
incubation
with
EBV-LCL,
CMV-pp65
and
Adv-
hexon/penton peptides respectively. In contrast, T-cells did not respond to CMVIE1, the
irrelevant peptide control.
Virus-specific CTL are expanded from the naïve CCR7/CD45RA+ T-cell population
We derived the virus specific CTLs described above from cord blood, which should lack
an EBV, CMV or Adv specific memory T-cell compartment. To determine whether the
virus-specific T-cells were indeed expanded from T-cells with a naïve phenotype (i.e.
CCR7+/CD45RA+ T-cell populations23), the mononuclear cells from 3 cord units (two
from CMV seronegative mothers and one from a CMV seropositive mother) were
incubated with antibodies for CD45RA and CCR7. We used flow cytometry to sort Tcells that were either double positive for both CD45RA and CCR7 or double negative for
CD45RA and CCR7 (Figure 2a). Each of these populations was stimulated with
transduced autologous antigen presenting cells and the virus specific activity evaluated
using IFNγ ELISPOT. After three stimulations, the phenotypes of the resultant CTL lines
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
showed a similar paucity of CD45RA+/CCR7+ T-cells (Figure 2b). As shown in Figure
2c T-cell lines derived from both CD45RA+/CCR7+ and CD45RA-/CCR7- populations
secreted IFN-γ in response to PMA-ionomycin. However, only T-cells derived from the
CD45RA and CCR7 double positive population secreted IFN-γ in response to EBV, CMV
and Adv antigen expressing target cells.
Virus-specific CTL derived from Cord Blood Recognize Immunodominant CD4
restricted Adv hexon epitopes
To map the adenovirus hexon epitope specificity of the CTL lines, we used an
overlapping peptide library representing the entire sequence of the Adv serotype 5
hexon protein
18;24
For the initial screening, CTL lines were stimulated by overnight
incubation with the 188 overlapping hexon peptides, divided into 11 pools, containing 17
or 18 peptides per pool.
The results from Adv hexon peptide pool screening of 6 evaluable CTL lines are
summarized in Table 2. Of the six CTL lines, three had reactivity against one adenovirus
hexon epitope and two lines had reactivity against multiple hexon epitopes. Three lines
had reactivity against adenovirus penton. The hexon epitopes identified in these cord
blood CTL lines were the same CD4-restricted epitopes as those identified in the multivirus CTL lines generated from adult peripheral blood.18
Figure 3 shows the details of such analyses from two of these cord blood derived CTL
lines. One line reacts predominantly against pool 1 (Figure 3a) and the other against
pool 10 (Figure 3b). Subsequent analysis with individual 20mer peptides contained in
these pools shows that the CTL reactivity in pool 1 could be mapped to overlapping
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
peptides
15
(aa
71-90
VDREDTAYSYKARFTLAVGD)
and
16
(aa
76-95
TAYSYKARFTLAVGDNRVLD) (Figure 3c), while CTL reactivity directed against pool 10
could be mapped to overlapping peptide 6 (aa 791-814 MYSFFRNFQPMSRQVVDDTK)
and peptide 7 (aa 796-815 RNFQPMSRQVVDDTKYKDYQ) (Figure 3d). Thus cord
blood derived CTLs have a similar pattern of epitope recognition to adult blood derived
CTLs. Furthermore, these epitopes are CD4-restricted with apparent immunodominance
since they have previously detected in multiple CTL lines derived from peripheral blood.
18
Virus-specific CTL derived from Cord Blood Recognizes unconventional CD4 and
CD8 restricted CMVpp65 epitopes
We characterized the CMVpp65 epitope specificity of the CTLs by incubating them with
CMVpp65 peptide pools
20
and measuring IFNγ release by ELISPOT assays. Table 3
illustrates that evaluable virus-specific lines derived from umbilical cord blood can
secrete IFNγ in response to several different CMVpp65 peptides, representing discrete
epitopes on the CMVpp65antigen.
A representative example is detailed in Figure 4.
This CTL line (HLA type:
A2,29;B35,44) had a detectable T-cell response to 20mer peptides from pools 7,
8,9,10,11,13,14 and 18 (Figure 4a). The 20mer peptides common to these three pools
were peptides 7, 8, 22, 23, 69 and 70 (Figure 4b). As shown in Figure 4c, screening of
these 20mer peptides revealed T-cells specific for at least two epitopes. They were the
known HLA A0201-restricted epitope MLNIPSINV (confirmed by tetramer as shown in
Figure
4d)
and
two
new
epitopes
(ALFFFDIDLLLQRGPQYSE
and
DTPVLPHETRLLQTGIHVRV) spanning the regions 347 to 358 and 31 to 51
respectively. By contrast, analysis of this CTL line for the known HLA A2 restricted
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
immunodominant CMVpp65 peptide (NLVPMVATV) was negative. Furthermore, 7/7 of
the CTL lines which were HLA A2 and/or A24 and/or B7 positive lacked T-cells specific
for the immunodominant A2 NLVPMVATV epitope and/or the B7 TPRVTGGGAM and
RPHERNGFTVL epitopes and/or the A24 QYDPVAALF epitope (Table 3). These results
are in contrast to CTL lines generated from peripheral blood donors11 with these HLA
types where 11/11 CTL lines exhibited specific activity against these immunodominant
epitopes (Table 4).
To characterize the EBV-specific T-cell response, we screened the CTL lines for known
immunodominant epitopes in EBNA 3, BZLF1 and LMP2.25 We found, however, no
evidence for such dominant-epitope specific T-cells (data not shown).
Virus-specific CTL derived from Cord Blood are expanded from Neonatal and not
Maternal T-cells
To confirm that the virus-specific T-cell responses observed in the cord blood derived
CTL lines were of neonatal and not maternal origin we used fluorescence in situ
hybridization (FISH) with sex chromosome-specific probes. X and Y chromosomes were
identified by red and green fluorescence, respectively (Figure 5). Of the 200
metaphases analyzed, 199 were positive for X and Y confirming that the cells were of
cord blood origin. For an additional three CTL lines from female neonates, the HLA types
of the mother were known. As shown in Table 1 the maternal HLA types were compared
to the CTL lines generated from cords C1000.4, C1001.4 and C1002.4. In all three, only
a single maternal HLA haplotype was found, so that the CTL line was derived from
neonatal not maternal T-cells.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
DISCUSSION
This study provides the first direct evidence that antigen-specific T-cells targeting
multiple viral epitopes from Adv, CMV and EBV can be primed simultaneously from CB
T-cells with a naïve phenotype. In addition, the multi-virus specific T-cells recognize an
array of epitopes after only 2 weeks expansion in vivo. Notably, the pattern of CMV
epitopes recognized appears to be different to “adult”-derived virus specific T-cells, while
the recognition of Adv epitopes is the same.
Park et al
26
have previously shown that CMV-specific T-cells can be generated in vitro
from umbilical cord blood, but the antigen and epitope specificity of the CMV-specific
CTL was largely uncharacterized. Moreover, it was unclear whether the CMV-specific
CTL they detected were derived from T-cells with a naïve CD45RA+/CCR7+
phenotype27. Questions also remained as to whether their CB-derived CTL came from
contaminating maternal blood cells. Our current study addresses these limitations and
also establishes the technology for simultaneously generating CTLs that are specific for
all three of the commonest viral infections after CBT, using a clinically relevant and GMP
compliant methodology.
Cord blood-derived T-cells can respond to endogenous antigens such as minor
histocompatibility antigens that are disparate between mother and fetus, and to
exogenous antigens such as parasites and environmental allergens.
28;29
They may also
respond to viral antigens if the mother is seropositive or has been vaccinated. Hence,
antigens in the maternal environment may prime fetal T-cells transplacentally and elicit
antigen-specific T-cell responses.30;31
In our study, however, it was evident that the
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
responses we obtained were derived by in vitro priming of the naive population of Tcells, and could be obtained irrespective of whether the mother was CMV seropositive or
consistently seronegative, pre and post term. We showed that these cells were not of
maternal origin and moreover, placental tissue from our donors was Adenovirus and
CMV negative by PCR analysis and by immunohistochemistry, further excluding the
possibility of priming in utero (data not shown).12
Although our ex vivo expanded cord blood derived multi-virus specific T-cells developed
a memory phenotype profile that was similar to peripheral blood derived multi-virus
specific T-cells11, their epitope specificity could be strikingly different. Thus, none of the
CB-derived CTL lines recognized the strongly immunodominant epitopes that are
universally identified in the peripheral blood CMVpp65 CTL lines generated from CMVseropositive donors (Table 4), with a preference instead for unconventional and even
novel CMVpp65 epitopes. Cord blood T-cells may be a transitional population between
thymocytes and adult T-cells28, so that the specificities we describe may thus represent
a “default response” by recent thymic emigrants, which have not undergone post natal
selection for more specific, and perhaps higher affinity anti-viral reactivity.22;32 This
hypothesis is supported by the broad Vβ repertoire we observed in cord blood-derived
CTL using spectratyping (data not shown). Murine studies revealing that promiscuous
low affinity TCR/MHC-peptide interactions on the surface of functionally immature
neonatal T-cells leads to an intensive “burst” of short-lived cellular immunity prior to the
establishment of conventional T-cell memory mediated by high affinity T-cells.33 Our own
human in vitro and the murine in vivo studies appear to reflect physiological events in
humans, since CMV infected fetuses are also deficient in CD8+ T-cells responding to the
canonical, immunodominant, B7-restricted RPH and A2- restricted NLV epitopes. 31
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Notwithstanding the recognition of non-canonical epitopes, cord blood-derived CMVspecific CTL are capable of specifically lysing CMVpp65-pulsed targets.
Moreover,
these usually "subdominant" CMV epitopes still successfully compete against Adv hexon
epitopes, since CTLs responding to CMV-pp65 continue to exceed in number the CTLs
responding to Adv hexon, a characteristic they share with memory-cell derived CTLs
directed to canonical pp65 epitopes.
While the specificity of cord blood naïve T-cell responses to CMV were markedly
different from adult seropositive peripheral blood T-cells, the responses of the two T-cell
populations to Adv virus were, perhaps surprisingly, indistinguishable. Using pools of
overlapping 20-mer peptides for hexon and pp65, we identified T-cells specific for the
same immunodominant CD4-restricted hexon epitopes found in adult seropositive
donors,
such
as
the
hexon
peptides
that
span
amino
acids
791–815
(MYSFFRNFQPMSRQVVDDTKYKDYQ).18 We do not know why cord and peripheral
blood T-cells should share epitope recognition for Adv but not CMV, but the difference
likely reflects the profoundly different biology of the two viruses. Adenovirus is an acute
infection that elicits strong innate immunity, followed by an adaptive CD4-restricted
hexon-specific T-cell response that in turn maximizes the range of epitopes recognized
by CD4+ and CD8+ effector cells.18 As a latent virus, however, CMV recruits a dominant
and oligoclonal CD8+ response.34 Cord blood T-cells are biased towards a Th2
phenotype,28 which may support development of a conventional adenoviral CD4-specific
T-cell response, but which in combination with the immaturity of the responding cord
blood T-cells, may redirect the CMV response away from the canonical epitopes.
Our explanation leads us to predict that our cord blood T-cell responses to EBV, a latent
virus like CMV, would also be to non-dominant epitopes. An evaluation for non-canonical
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
responses is more difficult for EBV than CMV, since T-cells from most responders
recognize epitopes from multiple latent and early lytic cycle EBV antigens. Nevertheless,
our screen of CTL lines for known immunodominant epitopes in EBNA 3, BZLF1 and
LMP225 failed to demonstrate any of the anticipated epitope specific T-cells (data not
shown) despite a functional cytokine and cytotoxic response to HLA-matched EBV-LCL
(Figures 1c and 1d) These observations are at least consistent with the differences we
propose between cord blood naive T-cell responses to acute and latent viral antigens.
Although we were able to generate multi-virus specific CTL from cord blood, success
was only achieved when we supplemented the cultures with IL-7, IL-12, and IL-15. Such
supplementation is unnecessary for the generation of virus specific CTLs from adult
memory T-cells,11 and may simply indicate a lack of memory T-cell precursors and/or a
deficiency of cord blood APC secretion of these cytokines. Neonatal dendritic cells
certainly appear to have a deficiency in IL-12 production, and we have shown how
cytokine supplementation can overcome these limitations.35 In the current studies, it is
possible that the altered epitope recognition we observed in the CMV responses are a
consequence of the addition of these supplementary cytokines. This seems unlikely as
the sole explanation for the phenomenon not only in view of the supporting data from
murine and human studies described above, but also because the responses to Adv
hexon epitopes were unchanged. Hence, we believe these differences more likely reflect
intrinsic differences in the responsiveness of naive CB versus memory T-cells derived
from CMV seropositive donors.
While antiviral pharmacotherapy may help prevent or treat CMV
36;37
and CD20 specific
antibodies may control EBV-associated lymphoproliferation,38 these drugs are
expensive, toxic and often ineffective due to primary or secondary resistance39.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Moreover, infections with adenoviruses (Adv) are increasingly reported after both
allogeneic HSCT and CBT and effective treatments are not currently available.37
Strategies to reduce the incidence of viral infections after CBT include using grafts with
the best HLA match and higher TNC, changes in conditioning and GvHD prophylaxis
regimens. However, adoptive transfer of peripheral blood derived T-cell lines enriched in
cells recognizing CMV, EBV and Adv can reproducibly control infections due to all three
viruses after allogeneic HSCT11 in a cost-effective manner. We suggest that our ability to
generate virus-specific CTL from cord blood against a plethora of epitopes recognized
by both CD4+ and CD8+ T-cells should minimize the risk of viral escape and maximize
therapeutic benefit on administration of these cells to cord blood recipients at risk of
severe viral disease.
AUTHORSHIP
Contribution: P.H and C.R.C. conducted the in vitro studies and contributed to the writing
of the paper; B.S, A.M.L, M.K, M.S and W.D helped develop the protocol and conduct
the in vitro studies. J.M. performed Vβ spectratyping analysis, H.L. provided statistical
support, A.P.G helped with scale up for Good Manufacturing Practice (GMP), C.M.R,
H.E.H. and G.D. helped with data analysis and provided advice on experimental design,
M.K.B. helped with data analysis and contributed to the writing of the paper, E.J.S
provided advice on ex vivo use of cord blood and contributed to the writing of the paper.
C.M.B developed the study, supervised the experiments and contributed to the writing of
the paper.
ACKNOWLEDGMENTS
This work was supported by a Dan L Duncan collaborative research grant to CMB and
EJS and by U54HL081007 from the NHLBI. AML is supported by an Amy Strelzer
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Manasevits Award from the National Marrow Donor Program, HEH by a Dan L Duncan
Chair, MKB by a Fayez Sarofim chair, CMB by a Leukemia and Lymphoma Society
Clinical Research Scholar award and EJS by a NCI RO1 CA061508-16.
CONFLICT OF INTEREST STATEMENT
The authors state that there are no conflicts of interest.
Reference List
1. Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of
hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med
1996;335:157-166.
2. Barker JN, Weisdorf DJ, DeFor TE et al. Transplantation of 2 partially HLAmatched umbilical cord blood units to enhance engraftment in adults with
hematologic malignancy. Blood 2005;105:1343-1347.
3. Komanduri KV, St John LS, de LM et al. Delayed immune reconstitution after cord
blood transplantation is characterized by impaired thymopoiesis and late memory
T-cell skewing. Blood 2007;110:4543-4551.
4. Walker CM, van Burik JA, De For TE, Weisdorf DJ. Cytomegalovirus infection after
allogeneic transplantation: comparison of cord blood with peripheral blood and
marrow graft sources. Biol.Blood Marrow Transplant. 2007;13:1106-1115.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
5. Matsumura T, Narimatsu H, Kami M et al. Cytomegalovirus infections following
umbilical cord blood transplantation using reduced intensity conditioning regimens
for adult patients. Biol.Blood Marrow Transplant. 2007;13:577-583.
6. Saavedra S, Sanz GF, Jarque I et al. Early infections in adult patients undergoing
unrelated donor cord blood transplantation. Bone Marrow Transplant. 2002;30:937943.
7. Szabolcs P, Niedzwiecki D. Immune reconstitution after unrelated cord blood
transplantation. Cytotherapy. 2007;9:111-122.
8. Boeckh M, Leisenring W, Riddell SR et al. Late cytomegalovirus disease and
mortality in recipients of allogeneic hematopoietic stem cell transplants: importance
of viral load and T-cell immunity. Blood 2003;101:407-414.
9. Brunstein CG, Weisdorf DJ, DeFor T et al. Marked increased risk of Epstein-Barr
virus-related complications with the addition of antithymocyte globulin to a
nonmyeloablative conditioning prior to unrelated umbilical cord blood
transplantation. Blood 2006;108:2874-2880.
10. Myers GD, Krance RA, Weiss H et al. Adenovirus infection rates in pediatric
recipients of alternate donor allogeneic bone marrow transplants receiving either
antithymocyte globulin (ATG) or alemtuzumab (Campath). Bone Marrow
Transplant. 2005;36:1001-1008.
11. Leen AM, Myers GD, Sili U et al. Monoculture-derived T lymphocytes specific for
multiple viruses expand and produce clinically relevant effects in
immunocompromised individuals. Nat.Med. 2006;12:1160-1166.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
12. Weinberg A, Enomoto L, Li S et al. Risk of transmission of herpesviruses through
cord blood transplantation. Biol.Blood Marrow Transplant. 2005;11:35-38.
13. Canto E, Rodriguez-Sanchez JL, Vidal S. Distinctive response of naive
lymphocytes from cord blood to primary activation via TCR. J.Leukoc.Biol.
2003;74:998-1007.
14. Chao NJ, Emerson SG, Weinberg KI. Stem cell transplantation (cord blood
transplants). Hematology.Am.Soc.Hematol.Educ.Program. 2004354-371.
15. Quintarelli C, Dotti G, De AB et al. Cytotoxic T lymphocytes directed to the
preferentially expressed antigen of melanoma (PRAME) target chronic myeloid
leukemia. Blood 2008;112:1876-1885.
16. Cohen Y, Scaradavou A, Stevens C et al. Factors affecting 100-day and 1-year
mortality following myeloablative single-unit cord blood transplantation in adults
and adolescents: a comprehensive meta-analysis of CIBMTR, NCBP and Eurocord
[abstract]. Biol Blood Marrow Transplant 2008;14:5.
17. Smith CA, Ng CYC, Heslop HE et al. Production of genetically modified EBVspecific cytotoxic T cells for adoptive transfer to patients at high risk of EBVassociated lymphoproliferative disease. J Hematother 1995;4:73-79.
18. Leen AM, Christin A, Khalil M et al. Identification of hexon-specific CD4 and CD8
T-cell epitopes for vaccine and immunotherapy. J.Virol. 2008;82:546-554.
19. Bollard CM, Aguilar L, Straathof KC et al. Cytotoxic T Lymphocyte Therapy for
Epstein-Barr Virus+ Hodgkin's Disease. J.Exp.Med. 2004;200:1623-1633.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
20. Kern F, Faulhaber N, Frommel C et al. Analysis of CD8 T cell reactivity to
cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides.
Eur J Immunol 2000;30:1676-1682.
21. Chang ST, Ghosh D, Kirschner DE, Linderman JJ. Peptide length-based prediction
of peptide-MHC class II binding. Bioinformatics. 2006;22:2761-2767.
22. Sili U, Huls MH, Davis AR et al. Large-scale expansion of dendritic cell-primed
polyclonal human cytotoxic T-lymphocytes using lymphoblastoid cell lines for
adoptive immunotherapy. J Immunother 2003;26:241-256.
23. Sallusto F, Lanzavecchia A. Mobilizing dendritic cells for tolerance, priming, and
chronic inflammation. J.Exp.Med. 1999;189:611-614.
24. Leen AM, Sili U, Vanin EF et al. Conserved CTL epitopes on the adenovirus hexon
protein expand subgroup cross-reactive and subgroup-specific CD8+ T cells. Blood
2004;104:2432-2440.
25. Khanna R, Burrows SR. Role of cytotoxic T lymphocytes in Epstein-Barr virusassociated diseases. Annu.Rev.Microbiol. 2000;54:19-48.
26. Park KD, Marti L, Kurtzberg J, Szabolcs P. In vitro priming and expansion of
cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes.
Blood 2006;108:1770-1773.
27. Parmar S, Robinson SN, Komanduri K et al. Ex vivo expanded umbilical cord blood
T cells maintain naive phenotype and TCR diversity. Cytotherapy. 2006;8:149-157.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
28. Thornton CA, Upham JW, Wikstrom ME et al. Functional maturation of
CD4+CD25+CTLA4+CD45RA+ T regulatory cells in human neonatal T cell
responses to environmental antigens/allergens. J.Immunol. 2004;173:3084-3092.
29. Mommaas B, Stegehuis-Kamp JA, van Halteren AG et al. Cord blood comprises
antigen-experienced T cells specific for maternal minor histocompatibility antigen
HA-1. Blood 2005;105:1823-1827.
30. Safdar A, Decker WK, Li S et al. De novo T-lymphocyte responses against
baculovirus-derived recombinant influenza virus hemagglutinin generated by a
naive umbilical cord blood model of dendritic cell vaccination. Vaccine
2009;27:1479-1484.
31. Pedron B, Guerin V, Jacquemard F et al. Comparison of CD8+ T Cell responses to
cytomegalovirus between human fetuses and their transmitter mothers.
J.Infect.Dis. 2007;196:1033-1043.
32. Micklethwaite K, Hansen A, Foster A et al. Ex vivo expansion and prophylactic
infusion of CMV-pp65 peptide-specific cytotoxic T-lymphocytes following allogeneic
hematopoietic stem cell transplantation. Biol Blood Marrow Transplant
2007;13:707-714.
33. Gavin MA, Bevan MJ. Increased peptide promiscuity provides a rationale for the
lack of N regions in the neonatal T cell repertoire. Immunity. 1995;3:793-800.
34. Day EK, Carmichael AJ, ten B, I et al. Rapid CD8+ T cell repertoire focusing and
selection of high-affinity clones into memory following primary infection with a
persistent human virus: human cytomegalovirus. J.Immunol. 2007;179:3203-3213.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
35. Cookson S, Reen D. IL-15 drives neonatal T cells to acquire CD56 and become
activated effector cells. Blood 2003;102:2195-2197.
36. Angarone M, Ison MG. Prevention and early treatment of opportunistic viral
infections in patients with leukemia and allogeneic stem cell transplantation
recipients. J.Natl.Compr.Canc.Netw. 2008;6:191-201.
37. Biron KK. Antiviral drugs for cytomegalovirus diseases. Antiviral Res. 2006;71:154163.
38. Kuehnle I, Huls MH, Liu Z et al. CD20 monoclonal antibody (rituximab) for therapy
of Epstein-Barr virus lymphoma after hemopoietic stem-cell transplantation. Blood
2000;95:1502-1505.
39. Avery RK. Update in management of ganciclovir-resistant cytomegalovirus
infection. Curr.Opin.Infect.Dis. 2008;21:433-437.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Table 1. Cord Blood and Maternal HLA Typing and CMV Serostatus
Cord ID
HLA typing Cord
Adv Hexon epitope
CB2
CB3
CB6
CB7
CB8
CB9
A2;24/B7
A2;29/B35;44
A2;33/B40(60);44
A11;30/B18;52
A2;3/B8;18
A29;31/B7;51
Penton specificity only
556-580: VPFHIQVPQKFFAIKNLLLLPGSYT
76-95: TAYSYKARFTLAVGDNRVLD
Hexon Pools 1,2,3,4 and penton specificity
Hexon Pools 6,10 and penton specificity
796-815:RNFQPMSRQVVDDTKYKDYQ
Table 2. Adenovirus Specificity of Cord Blood Virus-specific T-cell Lines
Cord ID
HLA typing Cord
HLA typing Mother
CB1
CB2
CB3
CB4
CB5
CB6
CB7
CB8
CB9
A2;3/B44;51
A2;24/B7
A2;29/B35;44
A3/B7;44
A3;29/B44;45
A2;33/B40(60);44
A11;30/B18;52
A2;3/B8;18
A29;31/B7;51
unknown
unknown
unknown
A2;3/B7;44
A2;3/B7;44
A2/B40(60);40(61)
unknown
unknown
unknown
CMV Serostatus of
Mother
negative
unknown
positive
positive
positive
negative
negative
negative
unknown
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Table 3. CMVpp65 Epitope Specificity of Cord Blood Virus-specific T-cell Lines
Cord
ID
HLA typing
Cord
CMVpp65 epitope(s)
CB1
A2;3/B44;51
CB2
A2;24/B7
31-51: DTPVLPHETRLLQTGIHVRV
126-146: INVHHYPSAAERKHRHLPVA
120-129: MLNIPSINV
351-371: LLQRGPQYSEHPTFT
CB3
A2;29/B35;44
CB4
A3/B7;44
CB5
CB6
CB7
A3;29/B44;45
A2;33/B40(60);
44
A11;30/B18;52
CB8
CB9
A2;3/B8;18
A29;31/B7;51
31-51: DTPVLPHETRLLQTGIHVRV
120-129: MLNIPSINV
347-358: ALFFFDIDLLL
Pools 14,16,19 (epitope not identified)
Pools 6,7,10,14,18 (epitope not identified)
221-241: DQYVKVYLESFCEDVPSGKL
256-276: MTRNPQPFMRPHERNGFTVL
11-31:MISVLGPISGHVLKAVFSRG
151-171:ASGKQMWQARLTVSGLAWTR
Not identified
116-131: LPLKMLNIPSINVHHYPSAA
Predicted Immunodominant
CMVpp65 epitope(s) not
identified
495–503: NLVPMVATV
495–503: NLVPMVATV
341–349: QYDPVAALF
417-426: TPRVTGGGAM
265-275: RPHERNGFTVL
495–503: NLVPMVATV
417-426: TPRVTGGGAM
265-275: RPHERNGFTVL
n/a
495–503: NLVPMVATV
n/a
495–503: NLVPMVATV
417-426: TPRVTGGGAM
265-275: RPHERNGFTVL
Table 4. CMVpp65 Epitope Specificity of Peripheral Blood Virus-specific T-cell
Lines
Peripheral Blood
CTL ID
PB1
HLA typing
CMVpp65 epitope(s)
A1;24/B51;57
PB2
A1;80/B7;53
PB3
PB4
PB5
PB6
PB7
PB8
A2/B8;49
A2402,3201/B1302,3501
A2;68/B35;40
A1;2/B8;15
A2,66/B27(w6),52(w6)
A2,3/B7,44
PB9
PB10
PB11
A2/B40(61);46
A2/B35;39
A2;3/B1501;5301
QYDPVAALF
369–379: FTSQYRIQGKL
113-121:VYALPLKML
TPRVTGGGAM
RPHERNGFTVL
NLVPMVATV
QYDPVAALF
NLVPMVATV
NLVPMVATV
NLVPMVATV
NLVPMVATV
TPRVTGGGAM
RPHERNGFTVL
NLVPMVATV
NLVPMVATV
NLVPMVATV
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
FIGURE LEGENDS
Figure 1. Expansion. immunophenotype and function of virus-specific cord bloodderived cytotoxic T lymphocytes. (a) For the generation of virus-specific CTL from
cord blood (virus-CTL), a total of 2x106 cord blood mononuclear cells (CBMC) were
activated with autologous Ad5f35CMVpp65-transduced dendritic cells (DC) in the
presence of IL-12, IL-7 and IL-15 followed by weekly stimulations with Ad5f35CMVpp65transduced LCL (LMP2-CTL) and twice weekly IL-2 feeds. Figure 1a represent CTL cell
numbers (x106) recorded from weekly cell counts comparing the expansion rates of 9
cord blood-derived CTL lines. (b) Reactivity of CTL lines (n = 9) with antibodies against
the T-cell and NK cell surface antigens and memory markers CD3, CD4, CD8, CD16,
CD56, TCRαβ, TCRγδ, CD45RA,CD28, CD62L, CD25, and the B cell surface antigen
CD19. (c)
51
Cr release at 4 h after coincubation of a representative CTL line with PHA
blasts pulsed with CMVpp65pepmix (CMVpp65 target), or PHA blasts pulsed with
CMVIE1pepmix (CMVIE1 target), or PHA blasts pulsed with Adenovirus hexon pepmix
(Advhexon target), nontransduced autologous EBV-LCL (EBV target), or allogeneic
EBV-LCL (allogeneic target). The data are percent lysis of targets by a representative
CTL line at E:T ratios of 40:1, 20:1, 10:1 and 5:1. (d) Virus-specific activity of all 9 CTL
lines after three stimulations as determined with the IFN-γ ELISPOT assay in response
to direct stimulation with CMVpp65pepmix (CMVpp65), CMVIE1pepmix (CMVIE1),
Adenovirus hexon pepmix (Adv hexon) or irradiated autologous EBV-LCL at an E:T ratio
of 4:1 (EBV). For each CTL line, mean values of triplicate experiments are reported.
SFC, spot-forming cells.
Figure 2. Virus-specific cytotoxic T lymphocytes from cord blood are derived from
the CD45RA+/CCR7+ naive T-cell population
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
(a)Using flow cytometry, T-cells from three cord blood units were gated on CD3+ cells
and then sorted for cells that were either double positive for both CD45RA and CCR7 or
double negative for CD45RA and CCR7. Each of these populations was then stimulated
with transduced autologous antigen presenting cells. (b)After three stimulations, the
phenotype of the CTL lines derived from cells that were either double positive for both
CD45RA and CCR7 (CD45RA+/CCR7+) or double negative for CD45RA and CCR7
(CD45RA-/CCR7-) was determined by flow cytometry. (c)Virus-specific activity of the
CTL lines was determined after three stimulations by the IFN-γ ELISPOT assay in
response to direct stimulation with CMVpp65pepmix (CMVpp65), CMVIE1pepmix
(CMVIE1), Adenovirus hexon pepmix (Adv hexon), irradiated autologous EBV-LCL at an
E:T ratio of 4:1 (EBV) or PMA-ionomycin (PMA-I). ELISPOT analysis of a T-cell line
derived from the CD45RA and CCR7 double positive fraction versus a T-cell line derived
from the double negative fraction from a representative cord blood unit from a CMV
seronegative mother is shown. Mean values (± s.d.) of triplicate experiments are
reported.
Figure 3. Specificity of Cord Blood CTL lines for Adenovirus hexon. Cord blood
derived CTL lines were screened by ELISPOT assay against 11 pools of hexon peptides
(20 mers overlapping by 15 aa) to identify which peptide pools were being recognized by
each CTL line. (a) The CTL line CB6 showed specific IFN-γ release specifically against
peptide pool 1. (b) The CTL line CB9 showed specific IFN-γ release specifically against
peptide pool 10. Results are expressed as spot-forming cells (SFC)/1x10e5 CTL. (c) To
identify the stimulating 20 mer peptides CTL line CB6 was re-screened against the
individual peptides contained in pool 1. The CTL reactivity against pool 1 mapped to the
overlapping
peptides
15
(VDREDTAYSYKARFTLAVGD)
and
16
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
(TAYSYKARFTLAVGDNRVLD). (d) In contrast, the reactivity in CTL line CB9 mapped
to peptides 6 (MYSFFRNFQPMSRQVVDDTK) and 7 (RNFQPMSRQVVDDTKYKDYQ)
in pool 10. Results are expressed as SFC/1x10e5 CTL.
Figure 4. Identification of CMVpp65 epitopes using a CMVpp65-peptide library
(a) CTLs (1x105/well) from CB3 (HLA-A2, A29, B35, B44) were stimulated with a
CMVpp65-peptide library pooled into 22 pools. Responses were measured in an 18-hour
IFN-γ ELISPOT assay. Shown is mean and standard deviation of duplicate wells. (b) All
peptides were divided into 22 pools in such a way that each peptide is present in 2
pools. This method allows determining those single peptides that likely induced
responses to the peptide pools. Thus, responses to pools 7, 8 and 13 or 9, 10 and 18 or
10, 11 and 14 may be induced by single peptides 7 and 8 or 69 and 70 or 22 and 23
respectively (c) Testing of these individual 20mers identifies the sequence of peptides 7
and 8 or 69 and 70 as most likely the overlapping 15 amino acids, as the CTL epitope. In
addition, the T-cell line mapped to a known HLA A2 restricted epitope (MLNIPSINV)
contained in peptides 23 and 24. (d) The polyclonal cord blood derived virus-specific
CTL line in which this epitope had been identified was stained with an HLA-A*0201
MLNIPSINV tetramer. Indicated is the percentage of tetramer positive cells within the
CD8+ population.
Figure 5. Virus-specific CTL derived from Cord Blood are of neonatal and not
maternal origin. To confirm the origin of the virus-specific T-cell responses observed in
the cord blood derived CTL line CB3 were of neonatal and not maternal origin we used
fluorescence in situ hybridization (FISH) with sex chromosome-specific probes. 200
metaphases were analyzed for X and Y chromosomes as identified by red and green
fluorescence, respectively.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Prepublished online May 14, 2009;
doi:10.1182/blood-2009-03-213256
Functionally active virus-specific T-cells that target CMV, adenovirus and
EBV can be expanded from naive T-cell populations in cord blood and will
target a range of viral epitopes
Patrick J. Hanley, Conrad Russell Young Cruz, Barbara Savoldo, Ann M Leen, Maja Stanojevic, Mariam
Khalil, William Decker, Jeffrey J Molldrem, Hao Liu, Adrian P Gee, Cliona M Rooney, Helen E Heslop,
Gianpietro Dotti, Malcolm K Brenner, Elizabeth J Shpall and Catherine M Bollard
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Advance online articles have been peer reviewed and accepted for publication but have not yet
appeared in the paper journal (edited, typeset versions may be posted when available prior to
final publication). Advance online articles are citable and establish publication priority; they are
indexed by PubMed from initial publication. Citations to Advance online articles must include
digital object identifier (DOIs) and date of initial publication.
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.