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TRANSPLANTATION
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,1,2 Conrad Russell Young Cruz,1 Barbara Savoldo,1,3 Ann M. Leen,1,3 Maja Stanojevic,1 Mariam Khalil,1
William Decker,4 Jeffrey J. Molldrem,4 Hao Liu,1,5 Adrian P. Gee,1,3 Cliona M. Rooney,1,3,6 Helen E. Heslop,1-3
Gianpietro Dotti,1,2,5 Malcolm K. Brenner,1-3 Elizabeth J. Shpall,4 and Catherine M. Bollard1-3,5
1Center
for Cell and Gene Therapy and Departments of 2Immunology and 3Pediatrics, Baylor College of Medicine, Texas Children’s Hospital and The Methodist
Hospital, Houston; 4Department of Stem Cell Transplantation and Cellular Therapy, University of Texas M. D. Anderson Cancer Center, Houston; and
Departments of 5Medicine and 6Virology, Baylor College of Medicine, Texas Children’s Hospital and The Methodist Hospital, Houston
The naive phenotype of cord blood (CB)
T cells may reduce graft-versus-host
disease after umbilical cord blood transplantation, 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 CD8ⴙ and CD4ⴙ T-cell subsets, predominantly of effector memory
phenotype. Each CTL culture also had
HLA-restricted virus-specific cytotoxic ef-
fector function against EBV, CMV, and
Adv targets. The CB CTLs recognized
multiple viral epitopes, including CD4restricted Adv-hexon 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
CB transplant recipients. This study is
registered at www.clinicaltrials.gov as
NCT00078533. (Blood. 2009;114:1958-1967)
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 nonwhites—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 patients who receive
an allotransplant.1,2
The major advantages of CB transplants compared with 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. Although a major disadvantage of CB is the low stem cell dose, which results in delayed
engraftment, there is also delayed immune recovery, particularly in
mismatched unrelated cord blood transplantations with the use of
antithymocyte globulin.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 rates of morbidity and
mortality.8-10 Although antiviral drugs can be beneficial for CMV and
EBV, effective agents are unavailable for Adv; 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 monoculturederived multivirus-specific cytotoxic T-lymphocytes (CTLs) from CMVseropositive donors can prevent and treat CMV, EBV, and adenoviral
disease after 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
naive phenotype, making them incapable of conferring protection
against CMV, EBV, and Adv during engraftment.3,12 Hence, the
development of virus-protective T-cell therapy for CB transplant
recipients requires the priming and extensive expansion of naive
T cells rather than the more limited and simple direct expansion of
a preexisting virus-specific memory T-cell population. This task is
made even more challenging because CB T cells have lower
cytotoxic activity and higher activation-induced cell death than do
peripheral blood–derived T cells.13,14 These limitations have prevented the successful generation of virus-specific CTLs in sufficient numbers for clinical use as prophylactic therapy of CMV,
EBV, and adenoviral infections after CB transplantation (CBT).
Submitted March 26, 2009; accepted May 7, 2009. Prepublished online as
Blood First Edition paper, May 14, 2009; DOI 10.1182/blood-2009-03-213256.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
© 2009 by The American Society of Hematology
1958
BLOOD, 27 AUGUST 2009 䡠 VOLUME 114, NUMBER 9
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BLOOD, 27 AUGUST 2009 䡠 VOLUME 114, NUMBER 9
VIRUS-SPECIFIC CYTOTOXIC T CELLS FROM CORD BLOOD
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Table 1. Cord blood and maternal HLA typing and CMV serostatus
Cord ID
HLA typing cord
CB1
A2;3/B44;51
Unknown
CB2
A2;24/B7
Unknown
Unknown
CB3
A2;29/B35;44
Unknown
Positive
CB4
A3/B7;44
A2;3/B7;44
Positive
CB5
A3;29/B44;45
A2;3/B7;44
Positive
CB6
A2;33/B40(60);44
A2/B40(60);40(61)
Negative
CB7
A11;30/B18;52
Unknown
Negative
CB8
A2;3/B8;18
Unknown
Negative
CB9
A29;31/B7;51
Unknown
Unknown
It has become evident that, in addition to antigen presentation
and intercellular costimulation, the ex vivo priming of antigenspecific 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 toward a central
memory phenotype, and influence the polarization of T cytotoxic
type 1 and T helper type 1 cells. In combination these cytokines augment
the generation of antigen-specific CTLs from naive 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 Adv even from the naive
T-cell population in human CB. Incorporation of EBV-transformed
B-lymphoblastoid cell lines in the antigen-presenting cells further
allows the 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.
Methods
CB units
Virus-specific CTLs were generated from CB units obtained frozen from
the M. D. Anderson Cancer Center Cord Blood Bank or fresh from mothers
who had consented to the protocol approved by the Institutional Review
Board (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 before use for the generation of CB-derived
CTLs, thereby mimicking the likely clinical setting. All CB samples 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 40 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 of 6 HLA class I (serologic) and II
(molecular) antigens. The unit must be cryopreserved in 2 fractions, with a
minimum of 2.5 ⫻ 107 total nucleated cells/kg prethaw 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 in
section entitled “Generation of multivirus-specific cultures derived from
umbilical CB.” 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 CB transplantation community.16
Generation of dendritic cells from cord blood
Umbilical CB was thawed and then purified by Ficoll (Lymphoprep;
Nycomed) gradient separation. CB mononuclear cells (CBMCs; 30 ⫻ 106)
were washed twice and resuspended in CellGenix media (CellGenix USA)
and plated at 5 ⫻ 106 cells/well in dendritic cell (DC) medium (CellGenix
media plus 2 mM L-glutamine; GlutaMAX; Invitrogen) in a 6-well plate
(Costar) for 2 hours at 37°C in a humidified CO2 incubator. Nonadherent
HLA typing mother
CMV serostatus of mother
Negative
cells were removed by rinsing with 1⫻ PBS (GibcoBRL) and refrozen.
Loosely adherent cells were cultured in DC media with 800 U/mL GM-CSF
(Sargramostim Leukine; Immunex) and 500 U/mL IL-4 (R&D Systems) for
7 days. IL-4 and GM-CSF were again added on day 3. On day 5, cells were
harvested for transduction.
Transduction of DCs
As our source of CMV and Adv antigens, we used a clinical-grade
recombinant Adv type 5 vector pseudotyped with an Adv type 35 fiber, that
encoded CMVpp65.11
On day 5, the CB-derived DCs were transduced with the clinical-grade
Ad5f35CMVpp65 vector11 at a multiplicity of infection of 10 IU/cell for
2 hours and matured in a cytokine cocktail of GM-CSF, IL-4, IL-1␤,
TNF-␣, IL-6, (R&D Systems) and PGE2 (Sigma-Aldrich) for 2 days. On
day 7, the DCs were harvested, irradiated (30 Gy) and then used to
stimulate virus-specific CTLs.
Generation and transduction of EBV-transformed B-cell lines
from umbilical CB
As the source of EBV antigens, 5 ⫻ 106 CBMCs were used for the
establishment of an EBV-transformed lymphoblastoid cell line (EBV-LCL),
by infection of CBMCs with concentrated clinical-grade supernatants from
a B95-8 working cell bank.17
Two days before CTL stimulation, LCLs were harvested, pelleted,
and incubated with Ad5f35CMVpp65 at a multiplicity of infection of
100 IU/cell for 90 minutes at 37°C. The cells were then resuspended at
5 ⫻ 105 cells/mL of complete media (RPMI [Hyclone] plus human serum
and GlutaMAX) and transferred to a 24-well plate at 2 mL/well and
cultured for 2 days before use as stimulator cells.
Generation of multivirus-specific cultures derived from
umbilical CB
Frozen CBMCs were thawed, washed, and resuspended in 45% RPMI
(Hyclone) and 45% CLICKS (Irvine Scientific) with 10% human serum
plus GlutaMAX-I (CTL medium). Cells were resuspended at 2 ⫻ 106/mL
and cocultured with autologous, irradiated transduced DCs at a ratio of
20 CBMCs to 1 DC in the presence of the cytokines IL-7, IL-12 (R&D
Systems), and IL-15 (CellGenix), all at 10 ng/mL. Cultures were restimulated on day 10 with irradiated Ad5f35CMVpp65-transduced LCL at a
responder-to-stimulator ratio of 4:1 plus IL-15 (10 ng/mL) and 1 week later
with irradiated autologous Ad5f35CMVpp65-transduced LCL at a responderto-stimulator ratio of 4:1. IL-2 (50-100 U/mL; Proleukin; Chiron) 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 with the use of flow cytometry and stimulated the DP and
DN populations with transduced DCs followed by transduced LCLs.
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, on
enrollment in our clinical trial of virus-specific T cells for the treatment of
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1960
BLOOD, 27 AUGUST 2009 䡠 VOLUME 114, NUMBER 9
HANLEY et al
Figure 1. Expansion, immunophenotype, and function
of virus-specific CB-derived cytotoxic T lymphocytes.
(A) For the generation of virus-specific CTLs from CB
(virus-CTLs), a total of 2 ⫻ 106 CB mononuclear cells (CBMCs) were activated with autologous Ad5f35CMVpp65transduced dendritic cells (DCs) in the presence of IL-12,
IL-7, and IL-15 followed by weekly stimulations with
Ad5f35CMVpp65-transduced LCL (LMP2-CTL) and twice
weekly IL-2 feeds. CTL cell numbers (⫻106) are recorded
from weekly cell counts comparing the expansion rates of
9 CB-derived CTL lines. (B) Reactivity of CTL lines (n ⫽ 9)
with antibodies against the T-cell and natural killer cell
surface antigens and memory markers CD3, CD4, CD8,
CD16, CD56, TCR␣␤, TCR␥␦, CD45RA, CD28, CD62L,
CD25, and the B-cell surface antigen CD19. Error bars
indicate mean ⫹ SD. (C) 51Cr release at 4 hours after
coincubation of a representative CTL line with PHA blasts
pulsed with CMVpp65 PepMix (CMVpp65 target), or PHA
blasts pulsed with CMVIE1 PepMix (CMVIE1 target), or PHA
blasts pulsed with Adv-hexon PepMix (Adv-hexon target),
nontransduced autologous EBV-LCL (EBV target), or allogeneic EBV-LCL (allogeneic target). The data are the percentage of lysis of targets by a representative CTL line at E/T of
40:1, 20:1, 10:1, and 5:1. (D) Virus-specific activity of all
9 CTL lines after 3 stimulations as determined with the IFN-␥
ELISPOT assay in response to direct stimulation with
CMVpp65 PepMix (CMVpp65), CMVIE1 PepMix (CMVIE1),
Adv-hexon PepMix (Adv hexon), or irradiated autologous
EBV-LCL at an E/T of 4:1 (EBV). For each CTL line, mean
values of triplicate experiments are reported. SFCs indicate
spot-forming cells.
viral infection after transplantation.11 All protocols were approved by the
Baylor College of Medicine IRBs 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, TCR␣␤, TCR␥␦, CD45RA, CD3/28, CD62L, CD4/25, CCR7
(Becton Dickinson). Samples were acquired on a FACScan flow cytometer
(Becton Dickinson), and the data were analyzed with the use of CellQuest
software (Becton Dickinson).
Enzyme-linked immunospot assay
Enzyme-linked immunospot (ELISPOT) analysis was used to determine the
frequency and function of T cells secreting IFN-␥ in response to PepMixes
(Jerini) 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 with flow cytometry) with CMV peptides.18
CBMCs stimulated with CMVIE1 PepMix and staphylococcal enterotoxin
B (1 ␮g/mL; Sigma-Aldrich) served as controls. Spot-forming cells were
enumerated by Zellnet Consulting and compared with input cell numbers to
obtain the frequency of virus-reactive T cells.
11 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 Adv-hexon peptide libraries
were designed to identify all possible HLA class I–restricted epitopes,
which have a length of 9 to 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 LCLs,22 PHA
blasts pulsed with CMVpp65 PepMix (Jerini), or Adv-hexon PepMix. As
control target cells we used unpulsed PHA blasts, PHA blasts pulsed with
irrelevant peptides, and HLA class I–mismatched LCLs. 51Cr-labeled target
cells were mixed with effector cells at doubling dilutions to produce the
effector-to-target (E/T) ratios specified. Target cells incubated in complete
medium or 5% Triton X-100 (Sigma-Aldrich) were used to determine
spontaneous and maximal 51Cr 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 plus or minus SDs are given unless
otherwise stated.
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.
Tetramer staining of CTLs (5 ⫻ 105) was previously described.19 For each
sample, 100 000 cells were analyzed using CellQuest software.
Panels of 20mer peptides (overlapping by 15 amino acids) covering the
entire amino acid sequence of CMVpp65 from the AD169 strain and
Adv-hexon from serotype 5 were synthesized.18,20 For CMVpp65,
22 peptide pools comprising 2 to 12 15mer peptides were prepared, so that
each 20mer peptide was represented in 2 pools.20 For Adv-hexon,
Results
Expanded CB-derived CTLs are virus specific and polyclonal
We prepared T-cell lines from 9 different CB units. After growth on
Ad5f35CMVpp65-transduced DCs and LCLs in the presence of IL-2,
IL-7, and IL-15, we harvested a median of 85.5 ⫻ 106 T cells (range,
60 ⫻ 106 to 2.65 ⫻ 108 T cells) by day 21 of culture. All of these cell
numbers would be sufficient for current T-cell adoptive transfer protocols, in which 1 ⫻ 107/m2 effectively reconstitute immunity to CMV,
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BLOOD, 27 AUGUST 2009 䡠 VOLUME 114, NUMBER 9
VIRUS-SPECIFIC CYTOTOXIC T CELLS FROM CORD BLOOD
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Figure 2. Virus-specific cytotoxic T lymphocytes from CB are derived from the CD45RAⴙ/CCR7ⴙ naive T-cell population. (A) With the use of flow cytometry, T cells
from 3 CB 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. Indicated are percentage of CD45RA⫺/CCR7⫺ T cells versus percentage
of CD45RA⫹/CCR7⫹ T cells that were selected. (B) After 3 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 3 stimulations by the IFN-␥ ELISPOT assay in response to direct stimulation with CMVpp65 PepMix (CMVpp65), CMVIE1 PepMix (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 CB unit from a CMV-seronegative mother is shown.
Mean values (⫾ SDs) of triplicate experiments are reported.
EBV, and Adv11 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 showed 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 IFN␥ ELISPOT assays to
discover whether the T-cell lines were indeed trivirus-specific
CTLs. Cytotoxic activity was tested against a panel of 51Cr-labeled
autologous and allogeneic target cells and showed that the CTL
lines killed PHA blasts only if they were pulsed with CMVpp65
PepMix or Adv-hexon PepMix (40% and 13%, respectively, at an
E/T of 20:1; Figure 1C). Furthermore, autologous EBV-LCLs were
also killed in this assay, showing an EBV-specific T-cell component. There was no cytotoxic activity against unpulsed PHA blasts,
PHA blasts pulsed with CMVIE1 PepMix (Figure 1C),or HLAmismatched EBV-LCLs (data not shown).
Similarly, Figure 1D shows that after 3 stimulations, virusspecific T-cell lines (n ⫽ 9) can secrete IFN␥ in response to EBV,
CMVpp65-, and Adv-hexon–expressing target cells with a mean
(⫾ SD) of 157 (⫾ 134), 209 (⫾ 210), and 74 (⫾ 65) spot-forming
cells per 105 T cells, after 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 CTLs are expanded from the naive
CCR7/CD45RAⴙ T-cell population
We derived the virus-specific CTLs from CB, 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 naive phenotype (ie, CCR7⫹/CD45RA⫹ T-cell populations23), the mononuclear cells from 3 cord units (2 from CMVseronegative mothers and 1 from a CMV-seropositive mother) were
incubated with antibodies for CD45RA and CCR7. We used flow
cytometry to sort T cells that were either DP for both CD45RA and
CCR7 or DN for CD45RA and CCR7 (Figure 2A). Each of these
populations was stimulated with transduced autologous antigenpresenting cells, and the virus-specific activity was evaluated with the
use of the IFN␥ ELISPOT. After 3 stimulations, the phenotypes of the
resultant CTL lines showed a similar paucity of CD45RA⫹/CCR7⫹
T cells (Figure 2B). As shown in Figure 2C, T-cell lines derived from
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Table 2. Adenovirus specificity of CB virus-specific T-cell lines
Cord
ID
HLA typing cord
CB2
A2;24/B7
Penton specificity only
CB3
A2;29/B35;44
556-580: VPFHIQVPQKFFAIKNLLLLPGSYT
CB6
A2;33/B40(60);44
76-95: TAYSYKARFTLAVGDNRVLD
CB7
A11;30/B18;52
Hexon pools 1, 2, 3, 4 and penton specificity
CB8
A2;3/B8;18
Hexon pools 6, 10 and penton specificity
CB9
A29;31/B7;51
796-815:RNFQPMSRQVVDDTKYKDYQ
Adv hexon epitope
both CD45RA⫹/CCR7⫹ and CD45RA⫺/CCR7⫺ populations secreted
IFN-␥ in response to PMA-ionomycin. However, only T cells derived
from the CD45RA and CCR7 DP population secreted IFN-␥ in response
to EBV, CMV, and Adv antigen-expressing target cells.
Virus-specific CTLs derived from CB recognize
immunodominant CD4-restricted Adv-hexon epitopes
To map the Adv-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 6 CTL lines, 3 had
reactivity against one Adv-hexon epitope, and 2 lines had reactivity
against multiple hexon epitopes. Three lines had reactivity against Adv
penton. The hexon epitopes identified in these CB 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 2 of these
CB-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 peptides 15 (aa 71-90 VDREDTAYSYKARFTLAVGD)
and 16 (aa 76-95 TAYSYKARFTLAVGDNRVLD; Figure 3C),
whereas 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, CB-derived CTLs have a similar pattern of
epitope recognition to adult blood–derived CTLs. Furthermore,
these epitopes are CD4-restricted with apparent immunodominance because they have been previously detected in multiple CTL
lines derived from peripheral blood.18
Virus-specific CTLs derived from CB recognizes
unconventional CD4- and CD8-restricted CMVpp65 epitopes
We characterized the CMVpp65 epitope specificity of the CTLs by
incubating them with CMVpp65 peptide pools20 and measuring
IFN␥ release by ELISPOT assays. Table 3 shows that evaluable
virus-specific lines derived from umbilical CB can secrete IFN␥ in
response to several different CMVpp65 peptides, representing
discrete epitopes on the CMVpp65 antigen.
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 3 pools were peptides 7,
8, 22, 23, 69, and 70 (Figure 4B). As shown in Figure 4C, screening
of these 20mer peptides showed T cells specific for at least
2 epitopes. They were the known HLA A0201-restricted epitope
MLNIPSINV (confirmed by tetramer as shown in Figure 4D) and
2 other epitopes (ALFFFDIDLLLQRGPQYSE and DTPVLPHET-
RLLQTGIHVRV) spanning the regions 347 to 358 and 31 to 51,
respectively. By contrast, analysis of this CTL line for the known
HLAA2–restricted immunodominant CMVpp65 peptide (NLVPMVATV) was negative. Furthermore, 7 of the 7 CTL lines that 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 in which 11 of 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 CTLs derived from CB are expanded from
neonatal and not maternal T cells
To confirm that the virus-specific T-cell responses observed in the
CB-derived CTL lines were of neonatal and not maternal origin we
used fluorescence in situ hybridization with sex chromosomespecific 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 chromosomes, confirming
that the cells were of CB origin. For an additional 3 CTL lines from
female neonates, the HLA types of the mother were known. As
shown in Table 1 the maternal HLA types were compared with the
CTL lines generated from cords C1000.4, C1001.4, and C1002.4.
In all 3, only a single maternal HLA haplotype was found, so that
the CTL line was derived from neonatal not maternal T cells.
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 naive
phenotype. In addition, the multivirus-specific T cells recognize an
array of epitopes after only 2 weeks of expansion in vivo. Notably,
the pattern of CMV epitopes recognized appears to be different to
“adult”-derived virus-specific T cells, whereas the recognition of
Adv epitopes is the same.
Park et al26 have previously shown that CMV-specific T cells
can be generated in vitro from umbilical CB, but the antigen and
epitope specificity of the CMV-specific CTL was largely uncharacterized. Moreover, it was unclear whether the CMV-specific CTLs
they detected were derived from T cells with a naive CD45RA⫹/
CCR7⫹ phenotype.27 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 3 of the commonest viral infections after CBT, using
a clinically relevant and GMP-compliant method.
CB-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
responses we obtained were derived by in vitro priming of the
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BLOOD, 27 AUGUST 2009 䡠 VOLUME 114, NUMBER 9
VIRUS-SPECIFIC CYTOTOXIC T CELLS FROM CORD BLOOD
1963
Figure 3. Specificity of CB CTL lines for adenovirus hexon. CB-derived CTL lines were screened by ELISPOT assay against 11 pools of hexon peptides (20mers
overlapping by 15 aa) to identify which peptide pools were being recognized by each CTL line. (A) The CTL line CB6 showed specific IFN-␥ release specifically against peptide
pool 1. (B) The CTL line CB9 showed specific IFN-␥ release specifically against peptide pool 10. Results are expressed as spot-forming cells (SFCs)/105 CTLs. (C) To identify
the stimulating 20mer peptides, CTL line CB6 was rescreened against the individual peptides contained in pool 1. The CTL reactivity against pool 1 were mapped to the
overlapping peptides 15 (VDREDTAYSYKARFTLAVGD) and 16 (TAYSYKARFTLAVGDNRVLD). (D) In contrast, the reactivity in CTL line CB9 was mapped to peptides 6
(MYSFFRNFQPMSRQVVDDTK) and 7 (RNFQPMSRQVVDDTKYKDYQ) in pool 10. Results are expressed as SFCs per 105 CTLs.
Table 3. CMVpp65 epitope specificity of cord blood virus-specific T-cell lines
Cord ID
HLA typing cord
CB1
A2;3/B44;51
CMVpp65 epitope(s)
31-51: DTPVLPHETRLLQTGIHVRV
Predicted immunodominant CMVpp65 epitope(s) not identified
495-503: NLVPMVATV
126-146: INVHHYPSAAERKHRHLPVA
CB2
A2;24/B7
120-129: MLNIPSINV
495-503: NLVPMVATV
351-371: LLQRGPQYSEHPTFT
341-349: QYDPVAALF
417-426: TPRVTGGGAM
265-275: RPHERNGFTVL
CB3
A2;29/B35;44
31-51: DTPVLPHETRLLQTGIHVRV
495-503: NLVPMVATV
120-129: MLNIPSINV
347-358: ALFFFDIDLLL
CB4
A3/B7;44
Pools 14,16,19 (epitope not identified)
417-426: TPRVTGGGAM
CB5
A3;29/B44;45
Pools 6,7,10,14,18 (epitope not identified)
N/A
CB6
A2;33/B40(60);44
221-241: DQYVKVYLESFCEDVPSGKL
495-503: NLVPMVATV
265-275: RPHERNGFTVL
256-276: MTRNPQPFMRPHERNGFTVL
CB7
A11;30/B18;52
11-31:MISVLGPISGHVLKAVFSRG
N/A
151-171:ASGKQMWQARLTVSGLAWTR
CB8
A2;3/B8;18
Not identified
495-503: NLVPMVATV
CB9
A29;31/B7;51
116-131: LPLKMLNIPSINVHHYPSAA
417-426: TPRVTGGGAM
265-275: RPHERNGFTVL
N/A indicates not applicable.
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BLOOD, 27 AUGUST 2009 䡠 VOLUME 114, NUMBER 9
HANLEY et al
Figure 4. Identification of CMVpp65 epitopes with the use of a CMVpp65-peptide library. (A) CTLs (105/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-␥ ELISPOT assay. Shown is mean and SD 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 probably 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 probably 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 CB-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.
Table 4. CMVpp65 epitope specificity of peripheral blood virus-specific T-cell lines
Peripheral blood CTL ID
HLA typing
CMVpp65 epitope(s)
PB1
A1;24/B51;57
QYDPVAALF 369-379: FTSQYRIQGKL 113-121:VYALPLKML
PB2
A1;80/B7;53
TPRVTGGGAM
PB3
A2/B8;49
NLVPMVATV
PB4
A2402,3201/B1302,3501
QYDPVAALF
PB5
A2;68/B35;40
NLVPMVATV
PB6
A1;2/B8;15
NLVPMVATV
PB7
A2,66/B27(w6),52(w6)
NLVPMVATV
PB8
A2,3/B7,44
NLVPMVATV
RPHERNGFTVL
TPRVTGGGAM
RPHERNGFTVL
PB9
A2/B40(61);46
NLVPMVATV
PB10
A2/B35;39
NLVPMVATV
PB11
A2;3/B1501;5301
NLVPMVATV
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BLOOD, 27 AUGUST 2009 䡠 VOLUME 114, NUMBER 9
VIRUS-SPECIFIC CYTOTOXIC T CELLS FROM CORD BLOOD
1965
Figure 5. Virus-specific CTL derived from CB are of
neonatal and not maternal origin. To confirm the origin of
the virus-specific T-cell responses observed in the CBderived CTL line CB3 were of neonatal and not maternal
origin we used fluorescence in situ hybridization (FISH) with
sex chromosome–specific probes. FISH probes specific to ␣
satellite centromeric region of chromosome X labeled with
spectrum orange and the satellite III (Yp12) region of chromosome Y labeled with spectrum green were obtained from
Abbott Laboratories. Hybridization was performed according
to the manufacturer’s protocols. The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and the
images were captured using 10⫻/25 aperture at magnification of 100⫻/1.40 oil R on Nikon E800 microscope equipped
with a cooled-charge coupled device (CCD) camera. The
cells were analyzed using Quips Pathvysion (Applied Imaging). A total of 200 interphase nuclei and 5 metaphase
spreads were analyzed for sex chromosome signal pattern.
naive population of T cells and could be obtained irrespective of
whether the mother was CMV seropositive or consistently seronegative, before and after term. We showed that these cells were not of
maternal origin; moreover, placental tissue from our donors was
Adv and CMV negative by polymerase chain reaction) analysis and
by immunohistochemistry, further excluding the possibility of
priming in utero (data not shown).12
Although our ex vivo–expanded CB-derived multivirus-specific
T cells developed a memory phenotype profile that was similar to
peripheral blood–derived multivirus-specific T cells,11 their epitope
specificity could be strikingly different. Thus, none of the CBderived CTL lines recognized the strongly immunodominant
epitopes that are universally identified in the peripheral blood
CMVpp65 CTL lines generated from CMV-seropositive donors
(Table 4), with a preference instead for unconventional and even
novel CMVpp65 epitopes. CB T cells may be a transitional
population between thymocytes and adult T cells,28 so that the
specificities we describe may thus represent a “default response” by
recent thymic emigrants, which have not undergone postnatal
selection for more specific and, perhaps, higher affinity antiviral
reactivity.22,32 This hypothesis is supported by the broad V␤
repertoire we observed in CB-derived CTLs with the use of
spectratyping (data not shown). Murine studies showing 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 before the
establishment of conventional T-cell memory mediated by highaffinity T cells.33 Our own human in vitro and the murine in vivo
studies appear to reflect physiologic events in humans, because
CMV-infected fetuses are also deficient in CD8⫹ T cells responding to the canonical, immunodominant, B7-restricted RPH and
A2-restricted NLV epitopes.31
Notwithstanding the recognition of noncanonical epitopes,
CB-derived CMV-specific CTLs are capable of specifically lysing
CMVpp65-pulsed targets. Moreover, these usually “subdominant”
CMV epitopes still successfully compete against Adv-hexon
epitopes, because 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.
Although the specificity of CB-naive T-cell responses to CMV
were markedly different from adult seropositive peripheral blood
T cells, the responses of the 2 T-cell populations to Adv were,
perhaps surprisingly, indistinguishable. With the use of pools of
overlapping 20mer 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 to 815 (MYSFFRNFQPMSRQVVDDTKYKDYQ).18 We do not know why CB and
peripheral blood T cells should share epitope recognition for Adv
but not CMV, but the difference probably reflects the profoundly
different biology of the 2 viruses. Adv is an acute infection that
elicits strong innate immunity, followed by an adaptive CD4restricted 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 CB T cells are biased toward a T helper
type 2 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 CB T cells,
may redirect the CMV response away from the canonical epitopes.
Our explanation leads us to predict that our CB T-cell responses
to EBV, a latent virus like CMV, would also be to nondominant
epitopes. An evaluation for noncanonical responses is more
difficult for EBV than for CMV, because 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 show any of the anticipated epitope-specific T cells (data
not shown) despite a functional cytokine and cytotoxic response to
HLA-matched EBV-LCLs (Figure 1C-D). These observations are
at least consistent with the differences we propose between CB
naive T-cell responses to acute and latent viral antigens.
Although we were able to generate multivirus-specific CTLs
from CB, 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 cells11 and may simply indicate a lack of memory T-cell
precursors a deficiency of CB APC secretion of these cytokines, or
both. Neonatal DCs 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 is a consequence of the addition of these supplementary
cytokines. This seems unlikely because the sole explanation for the
phenomenon not only in view of the supporting data from murine
and human studies described earlier 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 CMVseropositive donors.
Although antiviral pharmacotherapy may help prevent or treat
CMV36,37 and CD20-specific antibodies may control EBVassociated lymphoproliferation,38 these drugs are expensive, toxic,
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1966
BLOOD, 27 AUGUST 2009 䡠 VOLUME 114, NUMBER 9
HANLEY et al
and often ineffective because of primary or secondary resistance.39
Moreover, infections with adenoviruses 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 graft-versus-host
disease prophylaxis regimens. However, adoptive transfer of
peripheral blood–derived T-cell lines enriched in cells recognizing
CMV, EBV, and Adv can reproducibly control infections resulting
from all 3 viruses after allogeneic HSCT11 in a cost-effective
manner. We suggest that our ability to generate virus-specific CTLs
from CB 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 CB
recipients at risk of severe viral disease.
Acknowledgments
This work was supported by a Dan L. Duncan collaborative
research grant (C.M.B. and E.J.S.), the National Heart, Lung, and
Blood Institute (U54HL081007), an Amy Strelzer Manasevits
Award from the National Marrow Donor Program (A.M.L.), a Dan
L. Duncan chair (H.E.H.), a Fayez Sarofim chair (M.K.B.), a
Leukemia & Lymphoma Society Clinical Research Scholar award
(C.M.B.), and the National Cancer Institute (RO1 CA06150816; E.J.S.).
Authorship
Contribution: P.J.H. and C.R.Y.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.J.M. performed V␤ 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; and C.M.B. developed the
study, supervised the experiments, and contributed to the writing of
the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Catherine Bollard, Center for Cell and Gene
Therapy, Baylor College of Medicine, 6621 Fannin St, MC 3-3320,
Houston, TX 77030; e-mail: [email protected].
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2009 114: 1958-1967
doi:10.1182/blood-2009-03-213256 originally published
online May 14, 2009
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
Updated information and services can be found at:
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