Download Cytotoxic T-Lymphocyte–Defined Human Minor

From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Cytotoxic T-Lymphocyte–Defined Human Minor Histocompatibility Antigens
With a Restricted Tissue Distribution
By Edus H. Warren, Philip D. Greenberg, and Stanley R. Riddell
Cytotoxic T lymphocytes (CTL) specific for human minor
histocompatibility (H) antigens can be isolated from the
blood of major histocompatibility complex (MHC)-matched
allogeneic bone marrow transplant (BMT) recipients and
may play a prominent role in the graft-versus-host (GVH)
and graft-versus-leukemia (GVL) reactions (Tsoi et al, J
Immunol 125:2258, 1980; Tsoi et al, Transplant Proc 15:1484,
1983; Goulmy et al, Nature 302:159, 1983; Irle et al, Transplantation 40:329, 1985; and Niederwieser et al, Blood 81:2200,
1993). The identification of minor H antigens that are expressed in hematopoietic cells, including leukemic cells, but
not in fibroblasts and other tissue types has suggested that
such tissue-restricted antigens could potentially serve as
targets for T-cell immunotherapy to enhance GVL activity
without inducing GVH disease (de Bueger et al, J Immunol
149:1788, 1992; van der Harst et al, Blood 83:1060, 1994; and
Dolstra et al, J Immunol 158:560, 1997). To explore the
feasibility of this strategy, donor CD31CD81 CTL clones
specific for recipient minor H antigens were isolated and
characterized from allogeneic BMT recipients. CTL clones
were obtained from the majority of donor/recipient pairs.
Seventeen distinct minor H antigens distinguishable by their
MHC-restricting allele, population frequency, and/or distribution of tissue expression were defined by 56 CD31CD81 CTL
clones isolated from these patients. The MHC-restricting
alleles for these CTL clones included HLA-A2 and HLA-B7,
which had previously been shown to present minor H
antigens to CTL, as well as HLA-A3, -A11, -B8, -B53, and
-Cw7, which had not previously been described to present
minor H antigens to CTL. Estimated phenotype frequencies
for these 17 distinct minor H antigens range from 0.17 to
0.92. In vitro cytotoxicity assays using hematopoietic cells
and fibroblasts as target cells showed that 5 of the 17 minor
H antigens were expressed in both hematopoietic cells and
fibroblasts. However, 12 were presented for CTL recognition
only by hematopoietic cells and not by dermal fibroblasts
derived from the same donors. These results significantly
extend the spectrum of CTL-defined human minor H antigens that could potentially serve as target antigens for
cellular immunotherapy to promote GVL activity after allogeneic BMT.
r 1998 by The American Society of Hematology.
T
readily isolated and expanded ex vivo.24 In contrast, T cells
reactive with minor H antigens are present in low frequency in
the blood of unprimed donors and the isolation of minor H
antigen-specific T-cell clones from donor PBMC samples is
difficult.25,26 Goulmy et al27-29 overcame this obstacle and
generated polyclonal T-cell lines and several T-cell clones that
recognized recipient minor H antigens by obtaining PBMC
from the recipient after BMT, and stimulating these cells in vitro
with g-irradiated PBMC cryopreserved from the recipient
pretransplant.
These prior studies suggest the potential for adoptive T-cell
immunotherapy with minor H antigen-specific T-cell clones to
augment GVL reactivity after allogeneic BMT without causing
GVHD. However, only four CD81 cytotoxic T lymphocyte
(CTL)-defined minor H antigens that appear to be selectively
expressed by hematopoietic cells have been described: HA-1,
HA-2, and HA-5, which are all presented for T-cell recognition
by HLA-A2, and HB-1, which is presented by HLA-B44.28,30
Because 50% of BMT donor/recipient pairs do not express
HE USE OF T-CELL–depleted bone marrow (BM) for
major histocompatibility complex (MHC)-matched allogeneic BM transplantation (BMT) confers a reduced incidence
of graft-versus-host disease (GVHD) but a higher probability of
leukemic relapse compared with the use of unmodified BM.1-7
This observation and the results of experimental studies in
animal models have established a critical role for donor T
lymphocytes specific for recipient minor histocompatibility (H)
antigens in mediating the GVH and graft-versus-leukemia
(GVL) reactions that occur after allogeneic BMT and have
suggested that infusions of donor T cells may be useful
therapeutically in individuals at high risk of developing leukemic relapse after BMT.8 The adoptive transfer of donor
peripheral blood mononuclear cells (PBMC) containing large
numbers of CD31 T cells to patients with documented leukemic
relapse after allogeneic BMT has induced complete remissions
in most patients with relapse of chronic myelogenous leukemia
(CML) and some patients with relapse of acute myelogenous
leukemia (AML).9-22 Unfortunately, the administration of unselected polyclonal donor lymphocytes has also resulted in acute
and/or chronic GVHD in the majority of patients leading to
significant morbidity and mortality.9-22
A potential strategy to treat leukemic relapse without inducing GVHD would be to isolate donor T-cell clones specific for
recipient minor H antigens and to administer to the recipient
only those clones that recognize hematopoietic cells, including
leukemic blasts, but not nonhematopoietic tissues. The feasibility of using T-cell clones has been suggested by studies
demonstrating that cytomegalovirus (CMV)-specific T-cell immunity can be successfully reconstituted in allogeneic BMT
recipients without causing GVHD by the adoptive transfer of
donor T-cell clones selected for reactivity with CMV-infected
but not uninfected recipient cells.23,24 However, CMV seropositive donors maintain a high frequency of CMV-reactive T cells
in the PB, and T-cell clones specific for CMV antigens can be
Blood, Vol 91, No 6 (March 15), 1998: pp 2197-2207
From the Fred Hutchinson Cancer Research Center, Seattle, WA; and
the University of Washington, Seattle, WA.
Submitted June 24, 1997; accepted October 28, 1997.
Supported by grants from the National Institutes of Health (Grant No.
CA18029), the Lady Tata Memorial Trust (E.H.W.), and the Florence A.
Carter Fellowship from the American Medical Association-Education
and Research Fund (E.H.W.).
Address reprint requests to Edus H. Warren, MD, PhD, Program in
Immunology, M-758, Clinical Research Division, Fred Hutchinson
Cancer Research Center, 1124 Columbia St, Seattle, WA 98104.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9106-0010$3.00/0
2197
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2198
WARREN, GREENBERG, AND RIDDELL
HLA-A2 and 80% do not express HLA-B44, most recipients
would not be eligible for therapy targeting any of these four
minor H antigens.31 Moreover, even for donor/recipient pairs
expressing HLA-A2, the clinical use of HA-2 and HA-5 as
targets for GVL therapy is limited because HA-2 and HA-5 are
expressed in an estimated 95% and 7% of the population,
respectively.29 Thus, less than 10% of HLA-A21 donor/
recipient pairs would be appropriately discordant for the
expression of either of these antigens. HA-1 and HB-1 are
expressed in 69% and 28% of the population, respectively, and
recipients who express one of these antigens and who have a
donor that is discordant should be identified more frequently.29,30 CTL clones specific for HA-1 appear to recognize
leukemias of both myeloid and lymphoid lineages.30a However,
HB-1–specific CTL recognize only transformed B-lymphoid
cells and show no cytolytic activity against either monocytes or
phytohemagglutinin (PHA)-stimulated T cells, suggesting that
expression of HB-1 is restricted to the B-cell lineage.30 Thus,
adoptive immunotherapy with CD81 CTL specific for minor H
antigens as a general strategy to induce GVL activity after
allogeneic BMT will require the identification of additional
CD81 CTL-defined antigens that (1) exhibit restricted or
preferential expression in hematopoietic cells including myeloid and lymphoid leukemias and (2) are presented by class I
MHC molecules other than HLA-A2 and -B44.
To identify novel human minor H antigens that might be
potential targets for GVL therapy, we generated minor H
antigen-specific T cells from 10 allogeneic BMT donor/
recipient pairs. T-cell lines with recipient-specific reactivity
were obtained from 8 of the 10 cultures, and a panel of 56 CD31
CD81 CTL clones were isolated from 6 of these 8 T-cell lines.
Seventeen distinct minor H antigen specificities restricted by 7
different class I MHC alleles were identified using this panel of
CD81 CTL clones. CD81 CTL specific for 12 of these minor H
antigens lysed hematopoietic cells but not fibroblasts derived
from the same donors, and CTL specific for 6 of these
tissue-restricted antigens lysed leukemic blasts. These results
show that T cells potentially capable of mediating GVL activity
without causing GVHD can be isolated for use in adoptive
immunotherapy from a significant proportion of allogeneic
BMT recipients.
MATERIALS AND METHODS
Donor/recipient pairs. Ten patients with hematologic malignancies
undergoing allogeneic BMT and their HLA-matched related donors
were enrolled on this study. Characteristics of the 10 donor/recipient
pairs, including sex, HLA type, recipient’s diagnosis, source of hematopoietic stem cells, GVHD prophylaxis, and GVHD status, are shown in
Table 1. Nine of the 10 donor/recipient pairs were full siblings. Eight of
these nine sibling pairs (nos. 2, 3, 4, 5, 6, 8, 9, and 10 in Table 1) were
HLA-A-, -B-, -Cw-, -DR-, and -DQ-genotypically identical as demonstrated by serologic and DRB1 DNA sequence-based typing of the
siblings, the parents, and/or other siblings in each family. One pair (no.
7 in Table 1) was HLA-A-, -B-, -Cw-, and -DQ-identical by serology
but mismatched for one DRB1 allele (1501 v 1601). The remaining
donor/recipient pair (no. 1 in Table 1) was a mother/son combination
who were HLA-A- and -B-identical by serology but matched for only
one DR allele (DR 7, 6 v DR 7, 8).
Generation of Epstein-Barr virus (EBV)-transformed B-cell lines,
PHA blasts, and primary fibroblast lines. PB was obtained pretransplant from each donor and recipient to generate EBV-transformed
B-cell lines, and aliquots of PBMC were cryopreserved for later
preparation of PHA blasts. EBV-transformed B-cell lines (EBV-LCL)
were generated and cultured as described.32 Our laboratory has compiled a cell bank containing a large number of EBV-LCL lines generated
from individuals of known HLA type, and these were used in
experiments to define the MHC-restricting allele and the population
frequency for each of the minor H antigens. PHA blasts were generated
by culturing PBMC for 72 hours in CTL media containing 3 µg/mL
PHA (Sigma, St Louis, MO), washed and resuspended in CTL medium
supplemented with 25 U/mL recombinant human IL-2 (Chiron, Emeryville, CA), and used as target cells in cytotoxicity assays within 7 days.
Primary fibroblast lines were grown from explants of skin biopsy
specimens as described.33
Generation and characterization of minor histocompatibility antigenspecific T-cell lines. T-cell lines and clones were cultured in RPMIHEPES supplemented with 10% pooled, heat-inactivated human serum,
2 mmol/L L-glutamine, and 1% penicillin/streptomycin (termed CTL
medium). Donor T cells with reactivity for recipient minor H antigens
were generated in 24-well plates by stimulating in each well 1 to 4 3
106 responder PBMC obtained from the recipient posttransplant with 1
to 4 3 106 g-irradiated (35 Gy) PBMC obtained from the recipient
pretransplant. The cell lines were restimulated with g-irradiated recipient PBMC at 7 and 14 days after the initial stimulation and the media
was supplemented with interleukin-2 (10 to 15 U/mL) after each
restimulation. The resulting T-cell lines were expanded by restimulation
Table 1. Characteristics of the 10 Allogeneic BMT Donor/Recipient Pairs Recruited for This Study, Including Gender of Recipient and Donor,
Recipient’s Class I MHC Typing, Recipient’s Diagnosis, Source of Donor Hematopoietic Stem Cells, Regimen Used for GVHD Prophylaxis,
and Acute GVHD Status of the Recipient
Pair
No.
Recipient
Donor
HLA-A
HLA-C
HLA-B
1
2
3
4
5
6
7
8
9
10
Male
Female
Male
Male
Male
Male
Male
Female
Female
Male
Female
Male
Male
Female
Male
Male
Male
Male
Female
Female
2, 11
11, 24
1, 32
29, 30
3
2, 3
23, 2
3, 30
3, 24
2, 3
w6, w3
w3
w7, w1
w7, w6
w7
w5, w7
w7
w3, w4
w2
w4, w7
37, 62
51, 44
8, 27
8, 13
7
44, 35
58, 7
70, 53
51, 38
35, 7
Diagnosis
Source
of Donor
Stem Cells
GVHD
Prophylaxis
Acute
GVHD,
Grade
ALL
AML
MDS/AML
MDS/AML
MM
ALL
ALL
MDS/AML
AML
AML
BM
BM
BM
G-PBSC
G-PBSC
G-PBSC
BM
G-PBSC
G-PBSC
G-PBSC
ATG/MTX/CsA
MTX/CsA
MTX/CsA
MTX/CsA
MTX/CsA
MTX/CsA
ATG/MTX/CsA
MTX/CsA
MTX/CsA
MTX/CsA
III
II
III
I
III
II
III
I
I
II
Abbreviations: MDS/AML, AML arising in the setting of an antecedent myelodysplastic syndrome; MM, multiple myeloma; G-PBSC, granulocyte
colony-stimulating factor–mobilized PBSC; ATG, antithymocyte globulin; MTX, methotrexate; CsA, cyclosporine A.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
TISSUE-RESTRICTED MINOR H ANTIGENS
at weekly intervals with irradiated EBV-LCL derived from the recipient
pretransplant. After 4 to 6 weeks, the cultures were tested for cytolytic
activity against donor- and recipient-derived EBV-LCL and/or PHA
blast targets.
Cytotoxicity assays and blocking studies. Aliquots of 1 to 2 3 106
target cells were labeled with 50 µCi of 51Cr overnight, washed twice,
dispensed at 5 3 103 cells/well into triplicate cultures in 96-well
round-bottom plates, and incubated for 4 hours with effector cells at
various effector to target ratios in a total volume of 200 µL. Some assays
were performed by preincubating the target cells for 30 minutes at room
temperature in the presence and absence of 25 µg/mL of the anti-pan
class I MHC monoclonal antibody W6/32 (a generous gift of Dr Daniel
Geraghty, Fred Hutchinson Cancer Research Center, Seattle, WA). The
percentage of specific lysis was calculated using the standard formula.33
Isolation of minor H antigen-specific CD81 and CD41 T-cell clones.
T-cell lines exhibiting recipient-specific cytolytic activity were cloned
by limiting dilution in 96-well round-bottom plates. Each well received
200 µL of a cell suspension containing 5 3 104/mL g-irradiated (65 Gy)
recipient-derived EBV-LCL as antigen-presenting cells (APC), 2.5 3
105/mL g-irradiated (35 Gy) PBMC as feeder cells, and 2 cells/mL (0.4
cells/well) of responder T cells. In some experiments, CD41 T cells
were depleted from the T-cell lines before cloning by adherence to
tissue culture flasks coated with anti-CD4 monoclonal antibody (Applied Immune Sciences, Santa Clara, CA). After 13 to 14 days, wells
exhibiting T-cell growth were identified by microscopy and aliquots of
the well were screened for lytic activity against donor- and recipientderived EBV-LCL or PHA blast targets. T-cell clones with lytic activity
against only recipient-derived targets were expanded in vitro for further
analysis of phenotype and function.
Collection and processing of leukemic samples. Samples of PB
and/or BM were obtained from patients with acute myelogenous
leukemia (n 5 10), acute lymphoblastic leukemia (ALL; n 5 2), or
chronic lymphocytic leukemia (CLL; n 5 2), all of whom had either
primary refractory disease or relapse after conventional chemotherapy
or allogeneic BMT. All leukemic samples (PB and/or BM) contained
greater than 90% malignant cells as judged by morphologic criteria on
Wright-Giemsa–stained specimens. Leukemic cells were isolated by
Ficoll-Hypaque density gradient centrifugation. When not used immediately after isolation, the cells were cryopreserved in RPMI-HEPES with
20% human serum and 10% dimethyl sulfoxide for subsequent use.
PHA blast preparations from leukemic patients were prepared as
described above. Flow cytometry on these cell populations before use
showed that they consisted of greater than 90% CD31 cells.
Flow cytometric analysis of CTL clones and leukemic cells.
T-lymphocyte lines and clones were analyzed by two-color flow
cytometry for expression of CD3, CD4, and CD8 using fluorescein
Fig 1. Cytolytic activity of T-cell lines generated
from 10 donor/recipient pairs against recipient- and
donor-derived EBV-LCL targets. Lines were tested 4
to 6 weeks after initial stimulation for lytic activity
against recipient-derived (N) or donor-derived (M)
EBV-LCL targets in a standard 4-hour 51Cr release
assay at an effector to target (E:T) ratio of 20:1.
2199
isothiocyanate (FITC)-conjugated anti-CD3 and either phycoerythrin
(PE)-conjugated anti-CD4 or anti-CD8 (all from Becton Dickinson,
Mountain View, CA). Samples of leukemic blasts were analyzed for
expression of class I MHC by staining with the anti-pan class I MHC
monoclonal antibody W6/32 followed by FITC-conjugated goatantimouse Ig (Becton Dickinson). AML samples were also stained with
PE-conjugated anti-CD13 or anti-CD33 (Becton Dickinson) and ALL
and CLL samples were stained with FITC-conjugated anti-CD19
(Caltag, San Francisco, CA) or FITC-conjugated anti-CD20 (Becton
Dickinson). Analysis was performed on a FACScalibur flow cytometer
with CellQuest software (Becton Dickinson).
RESULTS
Cytotoxic minor H antigen-specific T-cell lines can be
generated from a majority of HLA-matched donor/recipient
pairs. To generate donor T cells reactive with recipient minor
H antigens, responder PBMC were isolated between 14 and 156
days after transplant from the PB of 10 transplant recipients
with donor engraftment, and cultured as described in the
Materials and Methods. Lines from 8 of the 10 donor/recipient
pairs exhibited preferential lytic activity against recipient
EBV-LCL compared with donor EBV-LCL (Fig 1). Three
cycles of stimulation with irradiated recipient PBMC were
generally required to first detect significant cytotoxicity against
recipient targets, and the cytotoxic activity of these polyclonal
T-cell lines was increased by restimulating the lines with
irradiated recipient-derived EBV-LCL for 2 or 3 additional
cycles (data not shown). In this small series of patients, the
ability to isolate recipient-specific cytolytic T cells did not
appear to correlate with the development of clinically significant GVHD. Recipient-specific cytolytic T-cell lines were
generated from 6 of the 7 patients with acute GVHD of grade II
or greater and from 2 of the 3 patients with mild (grade I)
GVHD.
Characterization of cell surface phenotype and isolation of
minor H antigen-specific cytotoxic T-cell clones. Analysis of
the cell surface phenotype of the 8 T-cell lines with preferential
lytic activity against recipient but not donor EBV-LCL showed
a mixed population of CD31CD41CD82 and CD31CD42CD81
cells in all cases (data not shown). To determine if CD81 T cells
contributed to the cytolytic activity against recipient target cells
and whether multiple minor H antigen specificities were being
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2200
WARREN, GREENBERG, AND RIDDELL
recognized by each line, T-cell clones were isolated in limiting
dilution cultures from the 8 T-cell lines using recipient EBVLCL as APC. In 5 of the cloning experiments (donor/recipient
pairs no. 1, 2, 3, 4, and 5; Table 1) the T cells were plated
without prior selection for CD41 or CD81 T cells. A total of 527
T-cell clones were isolated from 4 of these 5 T-cell lines.
Fifty-four of the T-cell clones exhibited cytolytic activity for
recipient but not donor EBV-LCL; 25 of these T-cell clones
were CD31CD41CD82 and 29 were CD31CD42CD81. Fourteen of the 25 cytolytic CD31CD41CD82 clones were derived
from donor/recipient pair no. 1, between whom a major
mismatch at DR (DR6 v DR8) was present, suggesting that
these clones may recognize MHC determinants rather than
minor H determinants. Because the focus of our study was to
identify minor H antigens presented by class I MHC molecules,
further characterization of the cytolytic CD31CD41CD82 clones
was not performed. Of the 29 CD31CD42CD81 CTL clones
isolated in these initial experiments, 1 was obtained from
donor/recipient pair no. 1, 2 from pair no. 2, 23 from pair no. 4,
and 3 from pair no. 5.
The small number of CD31CD42CD81 minor H antigenspecific CTL clones obtained from 3 of these 5 T-cell lines with
recipient-specific cytolytic activity suggested that enrichment
of CD81 T cells before cloning may be necessary to improve the
efficiency with which CD31CD81CD42 T-cell clones reactive
with recipient minor H antigens are isolated. Thus, 3 subsequent
T-cell lines derived from donor/recipient pairs no. 7, 8, and 10
(Table 1), respectively, were depleted of CD41 T cells and the
remaining cells plated at limiting dilution. A total of 357 T-cell
clones were isolated from 2 of these T-cell lines (pairs no. 7 and
8) and screened for cytolytic activity against recipient- but not
donor-derived EBV-transformed B-cell targets. Nine clones
from donor recipient/pair no. 7 and 18 clones from donor/
recipient pair no. 8 exhibited cytolytic activity against recipientbut not donor-derived target cells. These 27 minor H antigenspecific clones were all CD31CD42CD81.
Determination of class I MHC restriction for CD31CD81
CTL-defined human minor H antigens. Minor H antigens
recognized by CD81 CTL are presumed to be encoded by allelic
forms of polymorphic genes that differ in nucleotide sequence
between the donor and recipient and give rise to unique
antigenic peptide epitopes. Such CTL-defined minor H antigens
have previously been characterized by determining the class I
MHC-restricting allele and the frequency of the minor H
antigen in a population of individuals expressing this class I
MHC allele.29,34 To determine whether the minor H antigens
recognized by the CD81 CTL clones generated in our study
correspond to previously described minor antigens or represent
distinct specificities, the MHC-restricting elements for each of
the 56 CD81 CTL clones were identified by assessing the lytic
activity of the T-cell clones against a panel of EBV-transformed
B-cell lines derived from unrelated individuals, each of whom
shared only one class I MHC allele with the donor and recipient.
Seven different class I MHC alleles were identified to present
minor H antigens to the CTL clones isolated in this study. These
included HLA-A2 and HLA-B7, which were described in
earlier studies as restricting elements for minor H antigenspecific CTL, as well as HLA-A3, -A11, -B8, -B53, and -Cw7,
which have not previously been described as restricting alleles
for minor H antigen-specific CTL (Table 2). Representative data
identifying the class I MHC-restricting alleles for four of the
CTL clones are shown in Fig 2.
The proportion of individuals in the population that express
the gene encoding the minor H antigen can be estimated by
Table 2. Summary of the 17 Minor H Antigens Defined by the CD81 CTL Clones Described in This Report
Minor H
Antigen
Representative
Clone
Donor/ Recipient
Pair
Restricting
Element
Population
Frequency
Hematopoietic
Cells
Fibroblasts
A2-1
A2-2
A2-3
A2-4
A3-1
A11-1
A11-2
B7-1
B7-2
B7-3
B7-4
B7-5
B7-6
B8-1
B53-1
B53-2
Cw7-1
PAM-13
ATT-1
ATT-3
ATT-5
DRN-7
SJN-7
SJN-9
ATT-2
ATT-4
ATT-7
ATT-8
ATT-9
DRN-11
MRR-23
DJG-7
DJG-24
DRN-14
1
7
7
7
5
2
2
7
7
7
7
7
5
4
8
8
5
A2
A2
A2
A2
A3
A11
A11
B7
B7
B7
B7
B7
B7
B8
B53
B53
Cw7
0.70
0.47
0.28
0.17
0.74
(3/7)
(6/7)
0.31
0.92
0.85
0.77
0.60
0.56
0.50
(1/4)
(2/4)
0.70
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
1
1
2
2
2
2
1
2
2
2
2
2
Listed for each minor H antigen are a representative clone defining that minor H antigen, the class I MHC element that restricts recognition of the
minor H antigen, the donor/recipient pair from whom this clone was isolated, the estimated phenotype frequency of the minor H antigen in the
population bearing the restricting allele, and expression of the minor H antigen in hematopoietic cells and dermal fibroblasts (derived from single
donors), as inferred from in vitro cytotoxicity assays. For those clones for which fewer than 10 unrelated EBV-LCL targets bearing the appropriate
MHC restricting allele were available, the number of targets recognized/total number of targets tested is listed in parentheses in the place of the
population frequency. (1) Greater than 30% lysis at E:T 20:1. (2) Less than 5% lysis at E:T 20:1. All clones were tested against 2 or more types of
hematopoietic targets, including EBV-LCL, PHA blasts, and leukemic cells; a (1) appears if greater than 30% specific lysis at E:T 20:1 was
demonstrable against any one of these hematopoietic targets.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
TISSUE-RESTRICTED MINOR H ANTIGENS
2201
Fig 2. Identification of class I MHC-restricting elements for four representative CD81 minor H antigen-specific CTL clones. Each CTL clone was
assayed for lytic activity against a panel of EBV-LCL target cells derived from unrelated individuals each of whom shared only one class I MHC
allele with the donor/recipient pair from whom the clone was derived. Lytic activity at an E:T ratio of 20:1 against recipient-derived LCL,
donor-derived LCL, and LCL derived from unrelated individuals who shared the indicated HLA-A or HLA-B allele with the recipient and the donor
is plotted for (A) HLA-A3–restricted clone DRN-7, (B) HLA-B7–restricted clone DRN-11, (C) HLA-B8–restricted clone MRR-2, and (D) HLA-B53–
restricted clone DJG-24.
evaluating the lytic activity of each CTL clone against a large
panel of EBV-transformed B-cell targets bearing the relevant
class I MHC-restricting allele. For 36 of the 56 clones, an
estimate of the phenotype frequency of the minor H antigen in
the population was established by evaluating the lysis of
EBV-transformed B cells derived from at least 10 unrelated
individuals bearing the class I MHC-restricting allele. In some
cases, individual CTL clones that used the same class I
MHC-restricting element exhibited a different pattern of reactivity against the panel of unrelated EBV-LCL, indicating that
distinct minor H antigens were being recognized. For example,
four minor H antigen specificities presented by HLA-A2 and six
specificities presented by HLA-B7 could be identified by the
panel of CTL clones restricted by these alleles (Table 2). The
population frequencies of the four HLA-A2–restricted minor H
antigens were 0.17, 0.28, 0.47, and 0.70, respectively (Table
2).29 The population frequencies of the six HLA-B7–restricted
minor H antigens ranged from 0.31 to 0.92 (Table 2). Twentythree CD81 CTL clones were generated from donor/recipient
pair no. 4 and all recognized a minor H antigen presented by
HLA-B8. When these CTL were tested against a panel of 16
EBV-LCL derived from HLA-B81 donors, they lysed 10 of 10
EBV-LCL from male donors (.60% specific lysis) but 0 of 6
EBV LCL from female donors (,3% specific lysis), suggesting
that the minor H antigen recognized by these clones was
encoded or regulated by a gene on the Y chromosome.
The analysis of MHC restriction and population phenotype
frequency defined at least 17 distinct minor H antigenic
specificities, each of which was recognized by one or more of
the CD81 CTL clones generated in this study (Table 2).
Multiple CTL clones with identical MHC restriction and
patterns of reactivity against the panel of target cells were
frequently isolated from a single patient. However, none of the
17 minor H antigens identified in this study was recognized by T
cells derived from more than one patient (Table 2). Thus, this
analysis indicates that there is a large number of minor H
antigen disparities recognized by CD81 CTL and potentially
involved in GVH and GVL reactions after MHC-matched
sibling BMT and suggests that isolation of minor H antigenspecific T cells from additional donor/recipient pairs will
identify many new specificities.
Recognition of hematopoietic and nonhematopoietic cells by
CD31CD81 minor H antigen-specific CTL clones. To determine if any of the 17 minor H antigens identified by the CTL
isolated in this study were selectively presented by hematopoietic cells, CTL clones were tested for lytic activity against
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2202
recipient hematopoietic target cells including both EBV-LCL
and PHA blasts and against recipient dermal fibroblasts as a
representative nonhematopoietic target cell. CD81 CTL specific
for 5 of the 17 minor H antigens lysed the hematopoietic target
cells as well as fibroblasts (Table 2). Representative data for two
of these clones are shown in Fig 3A and B. However, CD81
CTL specific for the remaining 12 minor H antigens lysed only
the hematopoietic targets but not fibroblasts. Representative
data for two of these clones are shown in Fig 3C and D. CTL
clones recognizing these 12 minor H antigens with restricted
tissue expression were isolated from 4 of the 8 T-cell lines in
which T-cell cloning was performed and the class I MHCrestricting elements for these clones included HLA-A2, -A3,
-B7, -B8, -B53, and -Cw7 (Table 2).
Surprisingly, CD81 CTL clones recognizing the malespecific (H-Y) minor H antigen presented by HLA-B8 lysed
hematopoietic target cells but not dermal fibroblasts derived
from the same donors (Fig 4). This was not due to a failure of
these fibroblasts to present antigens or be lysed by CTL,
because a CTL clone specific for an HLA-A2–restricted minor
H antigen lysed the same fibroblasts as efficiently as hematopoietic cells (data not shown). Pretreatment of the male fibroblast
WARREN, GREENBERG, AND RIDDELL
targets with 500 U/mL interferon-g (IFN-g) for 48 or 72 hours
did not significantly sensitize them to lysis by any of these
HLA-B8–restricted clones (Fig 4). These results differ from
those obtained with the HLA-A1–, HLA-A2–, and HLA-B7–
restricted male-specific (H-Y) CTL clones described by other
investigators, which efficiently lysed both hematopoietic and
fibroblast target cells derived from male donors.28
Lysis of leukemic cells by CTL clones specific for tissuerestricted minor H antigens. The HLA-B53–restricted CD81
CTL clones isolated from donor/recipient pair no. 8 showed
significant lysis of leukemic cells from the recipient (data not
shown). To determine if the clones with limited tissue reactivity
derived from other recipients also lysed leukemic cells, samples
were obtained from 14 HLA-A31, HLA-B71, or HLA-B81
patients with primary refractory or relapsed AML (n 5 10),
ALL (n 5 2), or CLL (n 5 2). Flow cytometric analysis of the
PB and/or BM mononuclear cells obtained from the 10 individuals with AML showed that greater than 90% of the cells were
either CD131 and/or CD331 and PBMC from the 4 patients
with lymphoid leukemia contained greater than 90% CD191
and/or CD201 cells. The leukemic samples were stained for
surface expression of class I MHC with the monoclonal
Fig 3. Recognition of EBV-LCL, PHA blasts, and fibroblasts derived from single donors by four different CD81 minor H antigen-specific CTL
clones. EBV-LCL, PHA blasts, and fibroblasts derived from minor H antigen-positive individuals were used as targets in 51Cr release assays at E:T
20:1 for (A) HLA-A2–restricted clone PAM-13, (B) HLA-A2–restricted clone ATT-1, (C) HLA-A3–restricted clone DRN-7, and (D) HLA-B53–restricted
clone DJG-24.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
TISSUE-RESTRICTED MINOR H ANTIGENS
2203
Fig 4. The male-specific minor H antigen recognized by HLA-B8–restricted CTL clones is detected in
hematopoietic cells but not fibroblasts. Lytic activity
of a representative HLA-B8–restricted male-specific
CTL clone (MRR-23) against EBV-LCL (N), dermal
fibroblasts (£), and IFN-g–treated fibroblasts (500
U/mL for 48 hours; M) derived from four unrelated
HLA-B81 male donors. The effector to target ratio is
20:1.
antibody W6/32 to determine if complete or partial loss of class
I MHC might interfere with the presentation of minor H
antigens to CTL. None of the 14 leukemic samples contained a
significant population of class I MHClow or class I MHCnegative
cells (data not shown).
Four CTL clones which recognized distinct minor H antigens
presented by either HLA-A3, -B7, or -B8 were tested for their
ability to lyse leukemic cell targets from this panel in vitro. The
HLA-A3–restricted clone DRN-7 was assayed against 11
different HLA-A31 AML, ALL, and CLL samples. Significant
lytic activity was observed against 5 of 7 AML samples, 1 of 2
ALL samples, and 1 of 2 CLL samples, and this lytic activity
was inhibited in the presence of the anti-pan class I MHC
antibody W6/32 (Fig 5A). Clone DRN-7 was also tested for
lytic activity against PHA blast populations derived from the
same panel of leukemic patients, and lysed PHA blast targets
Fig 5. CD81 minor H antigenspecific CTL clones demonstrate
cytolytic activity against leukemic blasts in vitro that is blocked
by antibody to class I MHC. Activity of HLA-A3–restricted clone
DRN-7 (A) and HLA-B7–restricted
clones ATT-4 (B) and ATT-7 (C)
against panels of leukemic cells
in the absence (N) or presence
(M) of anti-pan class I MHC antibody W6/32 at 25 mg/mL. The
target cell panel in (A) was derived from 11 different HLA-A31
patients: 7 with AML, 2 with ALL,
and 2 with CLL. The target cell
panel in (B) and (C) was derived
from 5 different HLA-B71 patients: 4 with AML and 1 with
CLL. E:T was 20:1 in all experiments.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2204
WARREN, GREENBERG, AND RIDDELL
from the 7 patients against whom significant antileukemic
activity was also seen, but exhibited negligible lysis of PHA
blasts from the patients against whom no antileukemic activity
was demonstrable (data not shown).
The HLA-B7–restricted clones ATT-4 and ATT-7, which are
specific for minor H antigens present in 92% and 85% of the
population, respectively, were tested for lytic activity against 4
AML samples and 1 CLL sample. Both clones demonstrated
significant lytic activity against all of the leukemic targets, and
this lytic activity was significantly reduced in the presence of
the W6/32 antibody (Fig 5B and C). PHA blast targets derived
from the leukemic donors were also lysed (data not shown).
Three HLA-B8–restricted, H-Y–specific clones from donor/
recipient pair no. 4 were assayed against a panel of 2 male and 3
female HLA-B81 AML samples. Significant lytic activity was
seen against the male but not the female AML targets (data not
shown).
DISCUSSION
CD31CD81
The
class I MHC-restricted minor H antigenspecific CTL clones characterized in this study significantly
expand the spectrum of CTL-defined human minor H antigens.
Comparison of the results of class I MHC restriction, phenotype
frequencies, and distribution of tissue expression for the 17
minor H antigens identified here with those obtained for
previously described minor H antigens suggests that the antigens described here represent novel specificities.8,28-30 Similarity exists between two of the HLA-A2–restricted antigens
defined by clones ATT-3 and ATT-5 and the previously described HA-5 minor H antigen.28,29 These three minor H
antigens are all restricted by HLA-A2 and are detected in
hematopoietic cells but not fibroblasts. Clones ATT-3 and ATT-5
recognize distinct specificities as determined by differential
recognition of HLA-A21 target cells from the panel of unrelated
donors, but it is conceivable that one of these minor H antigens
could be identical to HA-5. The population frequency of 0.07
reported for HA-5 is lower than the frequencies of 0.17 and 0.28
we obtained for the minor H antigen defined by ATT-3 and
ATT-5, respectively, although this disparity could be due to the
different panel of EBV-LCL used in our analysis. HLA-B7–
restricted minor H antigens have also been described in
previous studies, but insufficient data were reported on the
frequency of these antigens in the population and their tissue
expression to determine if any correspond to one of the six
distinct HLA-B7–restricted minor H antigens defined by the
CTL clones generated and characterized in our study.34,35
The HLA-B8–restricted, male-specific H-Y antigen defined
by the CTL clones obtained from donor/recipient pair no. 4 is
distinguishable from H-Y antigens described by other investigators.28 CTL clones specific for the HLA-A2– and HLA-B7–
restricted H-Y antigens recognize epitopes derived from the
human homologue of the murine SMCY gene and lyse both
hematopoietic cells and fibroblasts.28,36,37 In contrast, the HLAB8–restricted H-Y antigen is expressed in hematopoietic cells
including EBV-transformed B cells, PHA blasts, and HLA-B81
AML blasts, but is not expressed sufficiently for CTL recognition in either untreated or IFN-g–treated fibroblasts. These
results suggest that the peptide epitope recognized by these
H-Y–specific clones is encoded by a gene distinct from SMCY
that is presumably located on the Y chromosome and whose
transcription, translation, and/or processing is regulated in a
tissue-specific fashion.
In the setting of MHC-matched allogeneic BMT, recipient
minor H antigens that are expressed on hematopoietic cells
including leukemic cells but are not widely distributed on
nonhematopoietic tissues could potentially be targets for adoptive therapy with donor-derived CTL clones to induce GVL
activity without causing GVHD. Twelve of the 17 CTL-defined
minor H antigens described in this report are detected in
recipient hematopoietic cells but not in skin fibroblasts. These
12 tissue-restricted antigens are presented by common class I
MHC alleles,31 including HLA-A2, -A3, -B7, -B8, and -Cw7,
and several are present in phenotype frequencies that suggest
that BMT donor/recipient pairs will often be discordant for one
or more of these minor H antigens. In the cohort of patients in
this study, CD31CD81 CTL clones recognizing tissuerestricted minor H antigens were isolated from 4 of the 8
donor/recipient pairs in whom T-cell cloning was attempted,
and it is conceivable that analysis of a larger number of clones
would identify such CTL in a higher fraction of patients. Two of
the 4 donor/recipient pairs from whom clones with tissuespecific reactivity were isolated (pairs no. 4 and 8) did not
develop clinically significant GVHD, in agreement with some38,39
but not all40 previous studies. CD81 CTL specific for 6 of the
tissue-restricted minor H antigens displayed lytic activity
against leukemic cells in vitro. CTL defining 4 distinct tissueresticted minor H antigens were tested against panels of
leukemic cells bearing the appropriate MHC-restricting allele
and were shown to lyse leukemias of both myeloid and
lymphoid phenotypes, demonstrating that, in addition to normal
hematopoietic cells such as T cells, B cells, and monocytes, the
antigens recognized are expressed in leukemic blasts. Thus, our
results significantly extend the number of minor H antigens that
could potentially be targeted with CD31CD81 CTL clones to
selectively induce GVL and demonstrate that a large proportion
of BMT patients would be candidates for adoptive T-cell
therapy.
A critical issue for the development of this approach to GVL
therapy is to demonstrate that expression of the genes encoding
candidate target minor H antigens is truly limited to hematopoietic cells. Dermal fibroblasts and keratinocytes have been used
as nonhematopoietic target cells because they can be readily
obtained from a skin biopsy and cultured in vitro. However,
defining the expression of minor H antigens in other tissues
using in vitro cytolytic assays is limited by the difficulties
inherent in obtaining and culturing samples from other tissue
sites. Moreover, data from such in vitro assays could underestimate the expression of minor antigens in tissues in vivo and thus
the potential for inducing or aggravating GVHD. Indeed, a
recent study identified a significant association of a mismatch of
the tissue-restricted HA-1 minor H antigen between donor and
recipient with the occurrence of grade II or higher GVHD in
adult BMT recipients.41 Thus, the identification of the genes
encoding minor H antigens is necessary to permit a comprehensive definition of gene expression in different tissues using
molecular techniques. Biochemical methods have been used to
isolate and sequence the peptide epitopes bound to MHC
molecules and this technology has recently been applied to
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
TISSUE-RESTRICTED MINOR H ANTIGENS
identify the sequence of human minor H antigen peptides.36,37,42
However, the short peptide sequence obtained with this approach, typically 8 to 11 amino acids in length, does not ensure
that the gene encoding the antigen will be identified in available
databases. A second approach to identifying genes encoding
CTL-defined antigens is based on cDNA expression cloning. In
this method, cDNA expression libraries are prepared from
antigen-positive cells and divided into small pools; these pools
are then cotransfected with a plasmid encoding the class I
restricting allele into antigen-negative target cells, and CTL are
used to screen the transfectants.43-46 This strategy has been used
to identify several genes encoding CTL-defined antigens expressed by melanoma cells and is being adapted in our
laboratory to identify genes encoding CTL-defined minor H
antigens.
The results of this and other studies have established that
minor H antigen-specific CTL clones are cytotoxic for leukemic
blasts in vitro, but the extent to which in vitro cytolytic activity
will correlate with in vivo antileukemic activity is unknown.
New insights into the biology of human AML underscore the
potential for the results of in vitro cytotoxicity assays to be
misleading. The transplantation of human AML cells into
NOD/SCID mice has revealed a hierarchy of cells in the
leukemic population with differing potential for establishing
leukemic engraftment.47,48 These studies have identified a
putative leukemic stem cell that is CD341, CD382, present in
exceedingly low frequency (,1 in 105 cells) in PB or BM
samples from AML patients, and capable of establishing
leukemic hematopoiesis in NOD/SCID mice.47,48 This suggests
that T cells used in immunotherapy of AML will have to
eliminate this rare AML stem cell. The activity of CD81 minor
H antigen-specific CTL clones against the putative AML stem
cell cannot easily be addressed with in vitro cytotoxicity assays
because of the rarity of this cell, but should be evaluable by
analyzing the effect of CTL on leukemic engraftment in the
NOD/SCID mouse. Preliminary studies have shown that CTL
clones generated in this study prevent engraftment of human
AML in the NOD/SCID model (Bonnet and Warren, manuscript
in preparation).
The results of this study suggest that there will be a large
number of distinct human minor H antigens that could be targets
for GVL therapy, and it may not be feasible in all circumstances
to pursue gene identification and studies of antileukemic
activity in the NOD/SCID mouse model. The ability to genetically modify human T-cell clones with the Herpes simplex virus
thymidine kinase gene to confer an inducible toxic phenotype
could permit the in vivo elimination of adoptively transferred T
cells if they caused severe GVHD.49-51 This strategy should
allow clinical evaluation of the antileukemic activity of minor H
antigen-specific T-cell clones for patients with relapse of AML
or ALL after allogeneic BMT.
ACKNOWLEDGMENT
The authors thank Jennifer Michaels for assistance in preparation of
the manuscript and Suzanne Xuereb for technical assistance with many
of the experiments described in this report.
2205
REFERENCES
1. Martin PJ, Hansen JA, Buckner CD, Sanders JE, Deeg HJ, Stewart
P, Appelbaum FR, Clift R, Fefer A, Witherspoon RP, Kennedy MS,
Sullivan KM, Fluornoy N, Storb R, Thomas ED: Effects of in vitro
depletion of T cells in HLA-identical allogeneic marrow grafts. Blood
66:664, 1985
2. Apperley JF, Jones L, Hale G, Waldmann H, Hows J, Rombos Y,
Tsatalas C, Marcus RE, Goolden AW, Gordon-Smith EC, Catovsky D,
Galton DAG, Goldman JM: Bone marrow transplantation for patients
with chronic myeloid leukemia: T-cell depletion with Campath-1
reduces the incidence of graft-versus-host disease but may increase the
risk of leukemic relapse. Bone Marrow Transplant 1:53, 1986
3. Mitsuyasu RT, Champlin RE, Gale RP, Ho WG, Lenarsky C,
Winston D, Selch M, Elashoff R, Giorgi JV, Wells J, Terasaki P, Billing
R, Feig S: Treatment of donor bone marrow with monoclonal anti-T-cell
antibody and complement for the prevention of graft-versus-host
disease. A prospective, randomized, double-blind trial. Ann Intern Med
105:20, 1986
4. Maraninchi D, Gluckman E, Blaise D, Guyotat D, Rio B, Pico JL,
Leblond V, Michallet M, Dreyfus F, Ifrah N, Bordigoni A: Impact of
T-cell depletion on outcome of allogeneic bone-marrow transplantation
for standard-risk leukaemias. Lancet 2:175, 1987
5. Goldman JM, Gale RP, Horowitz MM, Biggs JC, Champlin RE,
Gluckman E, Hoffmann RG, Jacobsen SJ, Marmont AM, McGlave PB,
Messner HA, Rimm AA, Rozman C, Speck B, Tura S, Weiner RS,
Bortin MM: Bone marrow transplantation for chronic myelogenous
leukemia in chronic phase. Increased risk for relapse associated with
T-cell depletion. Ann Intern Med 108:806, 1988
6. Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb
HJ, Rimm AA, Ringden O, Rozman C, Speck B, Truitt RL, Zwaan FE,
Bortin MM: Graft-versus-leukemia reactions after bone marrow transplantation. Blood 75:555, 1990
7. Ringden O, Remberger M, Aschan J, Lungman P, Lonnqvist B,
Markling L: Long-term follow-up of a randomized trial comparing T
cell depletion with a combination of methotrexate and cyclosporine in
adult leukemic marrow transplant recipients. Transplantation 58:887,
1994
8. Goulmy E: Human minor histocompatibility antigens. Curr Opin
Immunol 8:75, 1996
9. Kolb HJ, Mittermuller J, Clemm C, Holler E, Ledderose G, Brehm
G, Heim M, Wilmanns W: Donor leukocyte transfusions for treatment
of recurrent chronic myelogenous leukemia in marrow transplant
patients. Blood 76:2462, 1990
10. Cullis JO, Jiang YZ, Schwarer AP, Hughes TP, Barrett AJ,
Goldman JM: Donor leukocyte infusions for chronic myeloid leukemia
in relapse after allogeneic bone marrow transplantation [letter]. Blood
79:1379, 1992
11. Drobyski WR, Roth MS, Thibodeau SN, Gottschall JL: Molecular remission occurring after donor leukocyte infusions for the treatment
of relapsed chronic myelogenous leukemia after allogeneic bone
marrow transplantation. Bone Marrow Transplant 10:301, 1992
12. Bar BM, Schattenberg A, Mensink EJ, Geurts Van Kessel A,
Smetsers TF, Knops GH, Linders EH, De Witte T: Donor leukocyte
infusions for chronic myeloid leukemia relapsed after allogeneic bone
marrow transplantation. J Clin Oncol 11:513, 1993
13. Drobyski WR, Keever CA, Roth MS, Koethe S, Hanson G,
McFadden P, Gottschall JL, Ash RC, van Tuinen P, Horowitz MM,
Flomenberg N: Salvage immunotherapy using donor leukocyte infusions as treatment for relapsed chronic myelogenous leukemia after
allogeneic bone marrow transplantation: Efficacy and toxicity of a
defined T-cell dose. Blood 82:2310, 1993
14. Helg C, Roux E, Beris P, Cabrol C, Wacker P, Darbellay R, Wyss
M, Jeannet M, Chapuis B, Roosnek E: Adoptive immunotherapy for
recurrent CML after BMT. Bone Marrow Transplant 12:125, 1993
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2206
15. Hertenstein B, Wiesneth M, Novotny J, Bunjes D, Stefanic M,
Heinze B, Hubner G, Heimpel H, Arnold R: Interferon-alpha and donor
buffy coat transfusions for treatment of relapsed chronic myeloid
leukemia after allogeneic bone marrow transplantation. Transplantation
56:1114, 1993
16. Szer J, Grigg AP, Phillips GL, Sheridan WP: Donor leucocyte
infusions after chemotherapy for patients relapsing with acute leukaemia following allogeneic BMT. Bone Marrow Transplant 11:109, 1993
17. Porter DL, Roth MS, McGarigle C, Ferrara JL, Antin JH:
Induction of graft-versus-host disease as immunotherapy for relapsed
chronic myeloid leukemia. N Engl J Med 330:100, 1994
18. van Rhee F, Lin F, Cullis JO, Spencer A, Cross NC, Chase A,
Garicochea B, Bungey J, Barrett J, Goldman JM: Relapse of chronic
myeloid leukemia after allogeneic bone marrow transplant: The case for
giving donor leukocyte transfusions before the onset of hematologic
relapse. Blood 83:3377, 1994
19. Mackinnon S, Papadopoulos EB, Carabasi MH, Reich L, Collins
NH, Boulad F, Castro-Malaspina H, Childs BH, Gillio AP, Kernan NA,
Small TN, Young JW, O’Reilly RJ: Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid
leukemia after bone marrow transplantation: Separation of graft-versusleukemia responses from graft-versus-host disease. Blood 86:1261,
1995
20. Kolb HJ, Schattenberg A, Goldman JM, Hertenstein B, Jacobsen
N, Arcese W, Ljungman P, Ferrant A, Verdonck L, Niederwieser D,
Vanrhee F, Mittermueller J, Dewitte T, Holler E, Ansari H: Graft-versusleukemia effect of donor lymphocyte transfusions in marrow grafted
patients. Blood 86:2041, 1995
21. Giralt S, Hester J, Huh Y, Hirschginsberg C, Rondon G, Seong D,
Lee M, Gajewski J, Vanbesien K, Khouri I, Mehra R, Przepiorka D,
Korbling M, Talpaz M, Kantarjian H, Fischer H, Deisseroth A,
Champlin R: CD8-depleted donor lymphocyte infusion as treatment for
relapsed chronic myelogenous leukemia after allogeneic bone marrow
transplantation. Blood 86:4337, 1995
22. Collins RH Jr, Shpilberg O, Drobyski WR, Porter DL, Giralt S,
Champlin R, Goodman SA, Wolff SN, Hu W, Verfaillie C, List A,
Dalton W, Ognoskie N, Chetrit A, Antin JH, Nemunaitis J: Donor
leukocyte infusions in 140 patients with relapsed malignancy after
allogeneic bone marrow transplantation [see comments]. J Clin Oncol
15:433, 1997
23. Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME,
Greenberg PD: Restoration of viral immunity in immunodeficient
humans by the adoptive transfer of T cell clones. Science 257:238, 1992
24. Walter EA, Greenberg PD, Gilbert MJ, Finch RJ, Watanabe KS,
Thomas ED, Riddell SR: Reconstitution of cellular immunity against
CMV in recipients of allogeneic bone marrow by transfer of T-cell
clones from the donor. N Engl J Med 333:1038, 1995
25. Marijt WA, Veenhof WF, Brand A, Goulmy E, Fibbe WE,
Willemze R, van Rood JJ, Falkenburg JH: Minor histocompatibility
antigen-specific cytotoxic T cell lines, capable of lysing human
hematopoietic progenitor cells, can be generated in vitro by stimulation
with HLA-identical bone marrow cells. J Exp Med 173:101, 1991
26. van Lochem EG, Bakker A, Hoefsmit EC, de Gast GC, Goulmy
E: Analysis of dendritic-cell-induced primary T-cell responses between
HLA genotypically identical individuals. Hum Immunol 44:181, 1995
27. Goulmy E, Gratama JW, Blokland E, Zwaan FE, van Rood JJ: A
minor transplantation antigen detected by MHC-restricted cytotoxic T
lymphocytes during graft-versus-host disease. Nature 302:159, 1983
28. de Bueger M, Bakker A, Van Rood JJ, Van der Woude F, Goulmy
E: Tissue distribution of human minor histocompatibility antigens.
Ubiquitous versus restricted tissue distribution indicates heterogeneity
among human cytotoxic T lymphocyte-defined non-MHC antigens. J
Immunol 149:1788, 1992
29. van Els CA, D’Amaro J, Pool J, Blokland E, Bakker A, van Elsen
PJ, van Rood JJ, Goulmy E: Immunogenetics of human minor
WARREN, GREENBERG, AND RIDDELL
histocompatibility antigens: Their polymorphism and immunodominance. Immunogenetics 35:161, 1992
30. Dolstra H, Fredrix H, Preijers F, Goulmy E, Figdor CG, de Witte
TM, van de Wiel-van Kemenade E: Recognition of a B cell leukemiaassociated minor histocompatibility antigen by CTL. J Immunol
158:560, 1997
30a. van der Harst D, Goulmy E, Falkenburg JH, Kooij-Winkelaar
YM, van Luxemburg-Heijs SA, Goselink HM, Brand A: Recognition of
minor histocompatibility antigens on lymphocytic and myeloid leukemic cells by cytotoxic T-cell clones. Blood 83:1060, 1994
31. Imanishi T, Akaza T, Kimura A, Tokunaga K, Gojobori T: Alleles
and haplotype frequencies for HLA and complement loci in various
ethnic groups, in Tsuji K, Aizawa M, Sasasuki T (eds): HLA 1991:
Proceedings of the Eleventh International Histocompatibility Workshop
and Conference, vol 1. Oxford, UK, Oxford Science, 1992, p 1065
32. Rickinson AB, Rowe M, Hart IJ, Yao QY, Henderson LE, Rabin
H, Epstein MA: T-cell-mediated regression of ‘‘spontaneous’’ and of
Epstein-Barr virus-induced B-cell transformation in vitro: Studies with
cyclosporin A. Cell Immunol 87:646, 1984
33. Riddell SR, Rabin M, Geballe AP, Britt WJ, Greenberg PD: Class
I MHC-restricted cytotoxic T lymphocyte recognition of cells infected
with human cytomegalovirus does not require endogenous viral gene
expression. J Immunol 146:2795, 1991
34. Gubarev MI, Jenkin JC, Leppert MF, Buchanan GS, Otterud BE,
Guilbert DA, Beatty PG: Localization to chromosome 22 of a gene
encoding a human minor histocompatibility antigen. J Immunol 157:
5448, 1996
35. Marijt WA, Kernan NA, Diaz-Barrientos T, Veenhof WF, O’Reilly
RJ, Willemze R, Falkenburg JH: Multiple minor histocompatibility
antigen-specific cytotoxic T lymphocyte clones can be generated during
graft rejection after HLA-identical bone marrow transplantation. Bone
Marrow Transplant 16:125, 1995
36. Wang W, Meadows LR, den Haan JM, Sherman NE, Chen Y,
Blokland E, Shabanowitz J, Agulnik AI, Hendrickson RC, Bishop CE,
Hunt DF, Goulmy E, Engelhard VH: Human H-Y: A male-specific
histocompatibility antigen derived from the SMCY protein. Science
269:1588, 1995
37. Meadows L, Wang W, den Haan JM, Blokland E, Reinhardus C,
Drijfhout JW, Shabanowitz J, Pierce R, Agulnik AI, Bishop CE, Hunt
DF, Goulmy E, Engelhard VH: The HLA-A*0201-restricted H-Y
antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition. Immunity 6:273, 1997
38. van Els CA, Bakker A, Zwinderman AH, Zwaan FE, van Rood
JJ, Goulmy E: Effector mechanisms in graft-versus-host disease in
response to minor histocompatibility antigens. I. Absence of correlation
with cytotoxic effector cells. Transplantation 50:62, 1990
39. de Bueger M, Bakker A, Bontkes H, van Rood JJ, Goulmy E:
High frequencies of cytotoxic T cell precursors against minor histocompatibility antigens after HLA-identical BMT: Absence of correlation
with GVHD. Bone Marrow Transplant 11:363, 1993
40. Niederwieser D, Grassegger A, Aubock J, Herold M, Nachbaur
D, Rosenmayr A, Gachter A, Nussbaumer W, Gaggl S, Ritter M, Huber
C: Correlation of minor histocompatibility antigen-specific cytotoxic T
lymphocytes with graft-versus-host disease status and analyses of tissue
distribution of their target antigens. Blood 81:2200, 1993
41. Goulmy E, Schipper R, Pool J, Blokland E, Falkenburg F, Vossen
J, Gratwohl A, Vogelsang G, van Houwelingen H, van Rood J:
Mismatches of minor histocompatibility antigens between HLAidentical donors and recipients and the development of graft-versushost disease after bone marrow transplantation. N Engl J Med 334:281,
1996
42. den Haan JM, Sherman NE, Blokland E, Huczko E, Koning F,
Drijfhout JW, Skipper J, Shabanowitz J, Hunt DF, Engelhard VH,
Goulmy E: Identification of a graft versus host disease-associated
human minor histocompatibility antigen. Science 268:1476, 1995
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
TISSUE-RESTRICTED MINOR H ANTIGENS
43. Brichard V, Van Pel A, Wolfel T, Wolfel C, De Plaen E, Lethe B,
Coulie P, Boon T: The tyrosinase gene codes for an antigen recognized
by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp
Med 178:489, 1993
44. Coulie PG, Brichard V, Van Pel A, Wolfel T, Schneider J,
Traversari C, Mattei S, De Plaen E, Lurquin C, Szikora JP, Renauld JC,
Boon T: A new gene coding for a differentiation antigen recognized by
autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp
Med 180:35, 1994
45. Van den Eynde B, Peeters O, Debacker O, Gaugler B, Lucas S,
Boon T: A new family of genes coding for an antigen recognized by
autologous cytolytic T lymphocytes on a human melanoma. J Exp Med
182:689, 1995
46. Boel P, Wildmann C, Sensi ML, Brasseur R, Renauld JC, Coulie
P, Boon T, van der Bruggen P: BAGE: A new gene encoding an antigen
recognized on human melanomas by cytolytic T lymphocytes. Immunity 2:167, 1995
47. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, CaceresCortes J, Minden M, Paterson B, Caligiuri MA, Dick JE: A cell
2207
initiating human acute myeloid leukaemia after transplantation into
SCID mice. Nature 367:645, 1994
48. Bonnet D, Dick JE: Human acute myeloid leukemia is organized
as a hierarchy that originates from a primitive hematopoietic cell. Nat
Med 3:730, 1997
49. Lupton SD, Brunton LL, Kalberg VA, Overell RW: Dominant
positive and negative selection using a hygromycin phosphotransferasethymidine kinase fusion gene. Mol Cell Biol 11:3374, 1991
50. Riddell SR, Greenberg PD, Overell RW, Loughran TP, Gilbert
MJ, Lupton SD, Agosti J, Scheeler S, Coombs RW, Corey L: Phase I
study of cellular adoptive immunotherapy using genetically modified
CD81 HIV-specific T cells for HIV seropositive patients undergoing
allogeneic bone marrow transplant. The Fred Hutchinson Cancer
Research Center and the University of Washington School of Medicine,
Department of Medicine, Division of Oncology. Hum Gene Ther 3:319,
1992
51. Bonini C, Ferrari G, Verzeletti S, Servida P, Zappone E, Ruggieri
L, Ponzoni M, Rossini S, Mavilio F, Traversari C, Bordignon C:
HSV-TK gene transfer into donor lymphocytes for control of allogeneic
graft-versus-leukemia [see comments]. Science 276:1719, 1997
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1998 91: 2197-2207
Cytotoxic T-Lymphocyte−Defined Human Minor Histocompatibility
Antigens With a Restricted Tissue Distribution
Edus H. Warren, Philip D. Greenberg and Stanley R. Riddell
Updated information and services can be found at:
http://www.bloodjournal.org/content/91/6/2197.full.html
Articles on similar topics can be found in the following Blood collections
Transplantation (2228 articles)
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
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.